Antibody assay methods to assess risk for TIA/stroke

ABSTRACT

A methods, kits and compositions for diagnosing a central nervous system disorder, particularly transient ischemic attack or stroke, comprising measuring the level of NR2A and/or NR2B NMDA receptor or fragment thereof, in a biological sample from a human subject, and optionally measuring other biomarkers such as homocysteine and glutamate. The method is particularly useful for identifying individuals that are at risk for stroke, and for diagnosing stroke in an emergency room setting.

RELATION TO PRIOR APPLICATIONS

This application is a continuation of and claims priority to U.S.Utility Application Ser. No. 11/076,074, filed Mar. 3, 2005 (currentlypending), which is a continuation of U.S. Utility Application Ser. No.09/922,011, filed Aug. 2, 2001 (now issued as U.S. Pat. No. 6,896,872),which claims priority to U.S. Provisional Application No. 60/301,297,filed Jun. 7, 2001, and to U.S. Utility Application Ser. No. 09/632,749,filed Aug. 4, 2000, of which U.S. Ser. No. 09/922,011 was acontinuation-in-part.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the diagnosis, management andtherapy of central nervous system disorders such as stroke, transientishemic attack, and traumatic brain injury. In particular, the inventionrelates to methods and kits for evaluating these central nervous systemdisorders, in order to better respond to episodes of focal cerebralishemia, and to best manage the risk associated with future acuteincidences.

2. Background Information

Stroke or “brain attack” is clinically defined as a rapidly developingsyndrome of vascular origin that manifests itself in focal loss ofcerebral function. In more severe situations, the loss of cerebralfunction is global. A stroke occurs when the blood supply to the part ofthe brain is suddenly interrupted (ischemic) or when a blood vessel inthe brain bursts, spilling blood into the spaces surrounding the braincells (hemorrhagic). The symptoms of stroke are easy to spot: suddennumbness or weakness, especially on one side of the body; suddenconfusion or trouble speaking or understanding speech; sudden troubleseeing in one or both eyes; sudden trouble walking; dizziness; or lossof balance or coordination. (National Institute of NeurologicalDisorders and Stroke, 2001). Stroke is the most common devastatingneurologic disease in the world, and the third leading cause of death inworld after heart disease and cancer. Despite recent progressunderstanding stroke mechanisms, stroke management is still not optimalfor a number of reasons.

The importance of promptly diagnosing a stroke after symptoms appearcannot be overstated. Delays in diagnosis and medical interventionbeyond 3 hours after stroke onset may contribute to clinicaldeterioration and disability. An early diagnosis enables doctors to moreeffectively choose the emergency intervention such as anti-plateletor/and neuroprotective therapy, and also to make better prognoses ofdisease outcome. Successful treatment of stroke requires rapid statediagnosis, The delay in achieving an accurate and certain diagnosiswastes the limited amount of time available in which the brain canrespond to reperfusion, and significantly increases the risk ofhemmorrhage after most of the permanent injury has occurred (Marler J.R. Annl. Emergency Med. 1999, 33: 450-451).

Unfortunately, however, many people who have a stroke either do not seekimmediate medical care or suffer from delays in medical care even incountries where stroke care is advanced, such as the United States andEurope (Alberts M J, Hademenos G, Latchaw R E, et al. JAMA 2000;23:3102-3109). Several clinical criteria can be employed to diagnosewhether a patient is having a stroke, but even all these criteria do notalways allow one to differentiate the episode from other disorders, suchas epilepsy, syncope, and migraine (Toole J F. CerebrovascularDisorders. 1999. Lippincott, Williams & Wilkins, New York, 5^(th) Ed.,542 p). Moreover, progressing stroke is only partially predictable basedon clinical and neuroimaging data that is currently available toneurologists.

Transient ischemic attack (TIA) is a short-lived episode of focalneurologic deficit which often precedes the cerebral infarction of astroke. It occurs when the blood supply to part of the brain is brieflyinterrupted, and is typically accompanied by permanent brain damage(albeit less severe damage than normally results from a stroke). TIAsymptoms, which usually occur suddenly, are similar to those of strokebut do not last as long. Most symptoms of a TIA disappear within anhour, although they may persist for up to 24 hours. Symptoms caninclude: numbness or weakness in the face, arm, or leg, especially onone side of the body; confusion or difficulty in talking orunderstanding speech; trouble seeing in one or both eyes; and difficultywith walking, dizziness, or loss of balance and coordination. (NationalInstitute of Neurological Disorders and Stroke, 2001). Patients who havesuffered a TIA have 9.5 times greater risk of having a future strokethan those who have not had a TIA, and about one third of patients whosuffer a TIA will have an acute stroke in the future. (American StrokeAssociation, 2001). However, because the symptoms of TIA are short term,many patients do not recognize the event as a TIA or perceive the eventas a warning of a potentially impending stroke.

Standard treatments to reduce the risk of future stroke include the useof antiplatelet agents, particularly aspirin. People with atrialfibrillation (irregular beating of the heart) may be prescribedanticoagulants. The most important treatable factors linked to TIAs andstroke are high blood pressure, cigarette smoking, heart disease,carotid artery disease, diabetes, and heavy use of alcohol. Lifestylechanges can often be implemented to reduce these factors. However, it isnecessary to diagnose the TIA as a warning sign of impending strokebefore such treatments can be administered. Therefore, a laboratoryblood test to detect TIA or stroke, or the risk of suffering a TIA orstroke in the future, would be of tremendous benefit.

During the past 5 years a number of molecular and immunochemical assayshave been evaluated for clinical use in neurology. (Schenone A. et.al.Current Opinion in Neurology. 1999, 12: 603-604; Honnorat J. J. Neurol.Neurosurg. Psychiatry. 1996, 61:270-278). At present, the Thrombogene Vand two Thrombx tests are available for diagnosing stroke/thrombosisfrom Athena Diagnostic. These tests evaluate the frequent deep veinthrombosis and hypercoagulation states of patients to evaluate the needfor intravenous anticoagulant therapy. The Thrombogene V test detectsthe Factor V Leiden mutation by Polymerase Chain Reaction (PCR) in theblood of patients. The other two tests monitor changes of differentblood coagulation markers: antithrombin III protein C, factor IX, andanticardiolipin antibodies (IgG, IgM, IgA) by use of ELISA technique.These tests thus reveal the hypercoagulation state as a result of athrombotic events, such as stroke stroke.

Stroke can be related to different types of venous thromboembolisms,which are common disorders with considerable morbidity and potential formortality (Anderson, D.; Wells, P. Cur. Opinion in Hemat. 2000, 7:296-301). The biochemical marker: D-dimer, a breakdown product of across-linked fibrin blood clot that indicates the occurrence of plasminmediated lysis of cross-linked fibrin, has been extensively evaluatedfor use in diagnostic tests for indicating acute venous thromboembolism.Indeed, a fully automated, semi-quantative latex agglutination assaysthat uses turbimetric or agglutination endpoints has been developed thatprovides results within 20 minutes with sensitivity between 89% and 95%(Roussi J.; Bentolila L.; Contribution of D-dimer determination in theexclusion of deep venus thrombosis in spinal cord injury patients.Spinal Cord ,1999; v.37: p. 548-552). Unfortunately, however, thepresence of D-dimer may also be increased in other settings that resultin fibrin generation, including recent surgery, hemmorhage, trauma,cancer, and pregnancy (Anderson D R., Wells P S.; Thromb. Haemost.;1999; 82:878-886).

However, the foregoing tests do not elucidate upon the TIA/strokemechanisms that are actually responsible for the damage associated withneurotoxic molecular events. It is necessary to find out specific andsensitive biomarkers which could be helpful to recognize initial braindamage and which could help to choose not only the appropriateanticoagulant treatment, but also emergency or regular neuroprotectivetherapy.

It is well known that two of the three leading causes of death, namelycardiovascular diaseses and stroke, are the end result ofatherosclerosis. Thus, it is not surprising that several biochemicalmarkers implicated in thromboembolic processes are also reported to beassociated with stroke and, stroke risk. Among these are homocysteine,cholesterol and LDL (Cerebrovascular Disorders ed. by J. E. Toole.Lippincott Williams & Wilkins. 1999, pp. 34-35), which are alsoclassified as risk factors to cardiovascular and cerebrovasculardiseases. (Hankey G J., and Eikelboom J W. Lancet. 354: 407-413 (1999).Approximately one fourth of patients with symptomatic atherosclerosishave elevated plasma homocysteine levels caused by various factors. Highlevels of homocysteine may run in families with increased susceptibilityto heart attack and stroke (Graham I. J. Ir. Call. Phys. Surg. 1995; 24:25-30). Elevated plasma homocysteine may be a causal and modifiablerisk-factor for ischemic stroke, but the results of previous studieshave been conflicting (Deulofeu V N R, Chamorro A, Piera C. Med Clin(Barc). 1998; 110: 605-608; Yamamoto T, Rossi S, Stiefel M F, DoppenbergE, Zauner A, Bullock R, Marmarou A. Acta Neurochir. Suppl. (Wien) 1999;75:17-19).

The neurotoxic effect of excitatory amino acids (glutamate, aspartate)in the brain has also been well documented. The results of this workshow a correlation between glutamate content in the blood and theseverity of acute ischaemia (Castillo J, Dávalos A, Naveiro J, Noya M.Stroke 1996, 27:1060-1065; Castillo J, Dávalos A, Noya M. Lancet. 1997;349:79-83). Cerebral damage and its association with progressing strokehas been attributed to increased glutamate release, or low glutamatereuptake, both in animals and in humans (Dávalos A, Castillo J, SerenaJ, Noya M. Stroke 1997; 28:708-710).

However, only 56% of patients with progressing stroke are reported tohave high glutamate content in their blood serum (Dávalos A., Toni D.,Iweins F., et al., 1999, 30: 2631-2636). Moreover, even though glutamateis considered the strongest biochemical predictor of progressing stroke(Davalos A, and Castillo J. In Book: Cerebrovascular Disease. CurrentMed. Inc.: Philadelphia. 2000 Chapter 16, pp. 169-181), this markerremains non-specific for TIA. Glutamate changes have also been observedin the blood of patients with epilepsy and other nervous systemdisorders (Meldrum B S. J. Nutrition. 2000, 130:1007S-1015S).

Over the last three decades substantial progress has been made inelucidating the mechanisms by which cerebral ischemia leads to braindamage. The cellular and molecular mechanisms of cerebral ischemiaabnormalities have been better defined through the role of glutamate andglutamate receptors, one of the most distributed excitatoryneuroreceptors in brain, in regulating of initial stages of braindamage. Indeed, numerous molecular investigators consider glutamatereceptors to be one of the key biological receptors involved in themolecular mechanisms of TIA and stroke (Meldrum B S. J. Nutrition. 2000,130:1007S-1015S). According to a leading hypothesis, ischemia-inducedglutamate release activates these glutamate receptors. It has been shownthat glutamate and homocysteine (the sulfinic analog of aspartate)activate the glutamate binding site of NMDA receptors and participate inneurotoxic processes (Lipton S. A., Kim W. K., Choi Y. B., Kumar S., etal. PNAS. 1997, 94: 5923-5928).

Glutamate receptors are divided into two main groups: ionotropic andmetabotropic. The ionotropic neuroreceptors are ligand-gated ionchannels that are subdivided into NMDA, AMPA and kainate receptorsubtypes. There are four NR2 subunits: NR2A, NR2B, NR2C and NR2D, whichis responsible for Ca²⁺-permeability regulation. NMDA receptors can bemodified by ischemia, resulting in changes of ion permeability and/orion selectivity.

Recent research findings indicate that the blood of patients with CNSdisorders other than TIA or stroke exhibit properties ofautoimmunization to products of nerve cell degradation (Vincent A.,Oliver L., Pallace J. J Neuroimmun. 1999; 100: 169-180). For example, acorrelation between AMPA GluR1 autoantibodies and common epilepsy hasbeen shown (Dambinova et al. J. Neurol. Sci.1997; 152: 93-97; Dambinovaet al. J. Neurochem. 1998;71: 2088-2093), as has a correlation betweenAMPA GluR 3 autoantibodies and Rasmussen's encephalitis (Rogers S W,Andrews P I, Gahring L C, et al. Science. 1995;265:648-651; Twyman R E,Gahring L C, Spiess J, Rogers S W. Neuron. 1995; 14:755-762; Gahring LC, Twyman R I, Greenlee J E, Rogers S W. Mol. Med. 1995; 1:245-253).

In a similar vein, several researchers have reported an increase in NMDAreceptor synthesis, the appearance of high levels of receptor antigen,and the generation of autoantibodies to the receptors during the initialstages of cerebral ishemia (Gusev et al., J. Neurol. & Psych. 1996,5:68-72; Dambinova et al. J. Neurol. Sci. 1997, 152:93-97; Dambinova etal. J. Neurochem. 1998, 71: 2088-2093). Acting on this research, onecompany developed a laboratory kit (cerebral ischaemia (CIS)-test) thatdetects autoantibodies to the N-terminus domain of the NR2A subunit inthe blood of patients with TIA or stroke (Gusev E. I., Skvortsova V I,Alekseev A A, Izykenova G A, Dambinova S A. S.S Korsakov's J. Neurol. &Psych. 1996; 5:68-72). The N-terminus domain of the NR2A subunit of NMDAreceptors was selected as the immunoreactive epitope on the basis ofmolecular biological and experimental studies showing that this epitopeis the most immunoreactive region of the receptor (Dambinova S A,Izykenova G A. J. High Nervous Activity. 1997; 47:439-446).

More recently, researchers have reported a correlation between theeffectiveness of a stroke treatment regimen and the levels ofautoantibodies to the NR2A and NR2B subunits of NMDA. In particular,these researchers have reported increased titers of autoantibodies tothe NR2A and NR2B subunits of NMDA in the blood of patients severelyaffected by stroke, and a reduction of the autoantibodies, accompaniedby an improvement in neurological function, during therapy by glycine—anon-specific agonist of NMDA receptors (Gusev et.al. CerebrovascularDiseases. 2000, 10: 49-60). Patients that responded positively toglycine had lower autoantibody titers than patients who were nottreated, and had levels of autoantibodies that were close to the levelsof autoantibodies in control subjects.

Unfortunately, the use of NR2A and NR2B autoantibodies in the diagnosisof stroke or TIA does not provide a real-time assessment of the damagebeing done by a stroke or TIA. Rather, because of the time the immunesystem requires to mount an immune response, and to generate NR2A andNR2B autoantibodies, methods that test for these antibodies at bestprovide a delayed assessment of the extent and severity of stroke orTIA.

Investigators from Canada (Hill M. D., Jackowski G., Bayer N., LawrenceM., Jaeschke R. Can. Med. Assoc. J. 2000, 163: 1139-1140) have proposeda new diagnostic laboratory assay for differentiating stroke subtype.They designed a preliminary prospective cohort study to test a panel ofbiochemical markers (neuron-specific enolase[NSE], myelin basic protein[MBP], S-100 [betta] protein and thrombomodulin [Tm]) in blood samplesfrom patients with acute ischemic stroke. These markers were chosenbecause they cover important cellular components of the brain that mightbe damaged in acute stroke. The 4 biochemical markers were assayed usinga standard ELISA technique.

The results of this investigation demonstrated elevated levels of NSE in89% of the patients admitted in hospitals, Tm in 43%, MBP in 39% andS-100 [beta] in 32%. At least one of the markers was elevated onadmission in 93% of the acute stroke patients. By stroke type, 100% ofthe patients with lacunar stroke, 100% of those with posteriorcirculation stroke and 90% of those with partial anterior circulationstroke had elevated NSE levels on admission. Conversely, none of thepatients with lacunar stroke had en elevated S-100[beta] level initiallyor subsequently. Peak levels of NSE, S-100 [beta] and MBP, but not ofTm, were significantly correlated with admission NIHSS scores (p<0.05).

For stroke, 3 hours is an outside limit for administering appropriatetherapies. The focus must change from extensive evaluation before anyaction to a well-planned acute emergency therapy developed using anappropriate diagnostic strategy. Every future advance to improve theoutcome after TIA/stroke will depend on a fast initial response—withinminutes and not hours (Marler J. R. Annl. Emergency Med. 1999, 33:450-451). Therefore, it is especially important to develop a fast andsimple method (within one hour) of detecting brain and blood biomarkerscapable of recognizing the initial processes of TIA/stroke beforeirreperable ischemic damage ensues.

OBJECTS OF INVENTION

Therefore, it is an object of the invention to provide biochemicalmethods and kits for diagnosing central nervous system disorders such asTIA and stroke.

It is another object of the present invention to improve upon theaccuracy of currently available methods for diagnosing TIA and stroke,and to more accurately diagnose TIA and stroke to the exclusion of othernervous system disorders or traumatic brain injury.

It is still another object of the present invention to provide methodsof diagnosing stroke using biochemical markers that distinguish betweenhemorrhagic and ischemic stroke.

Still another object of the invention is to provide biochemical analysesof the extent and progression of TIA or stroke, or the infarctionresulting from the TIA or stroke.

It is another object of the present invention to provide rapidbiochemical methods and kits for diagnosing TIA and stroke, to providereal-time assessments of TIA or stroke, within a window of time thatpermits effective therapeutic intervention.

It is another object of the present invention to provide rapid andinexpensive biochemical methods and kits for diagnosing TIA and stroke,which can be performed at frequent intervals to monitor the progressionof a TIA or stroke, or the effectiveness of therapy administered againstTIA or stroke.

Still another object of the present invention is to provide diagnosticmethods and kits for assessing the risk of incurring a TIA or stroke,and for monitoring the remission of risk factors for TIA or stroke.

Still another object of the invention is to provide a panel of rapidmultiple panel of biomarkers for assessing the nature, severity andprogression of TIA or stroke, and thereby to enable a more effectiveselection of intervention therapy.

SUMMARY OF THE INVENTION

It has unexpectedly been discovered that levels of circulating NMDAreceptor proteins or fragments thereof can be assessed using diagnostickits and processes, and that levels of these proteins or fragments canbe used to clinically evaluate patients suffering from ishemic centralnervous system disorders such as stroke or TIA. When analyzed incombination with other biomarkers for stroke and TIA, such as thethromboembolic marker homocysteine, or the excititory amino acidglutamate, these proteins can diagnose the existence of a stroke withremarkable accuracy (generally greater than 89%). In contrast, theefficacy of single parameters for early diagnosis of stroke is 58% forglutamate, 66% for homocysteine, and 79% for NMDA receptors. The rapidevaluation of these neural ischemic biomarkers in an emergency roomsetting will greatly enhance the confidence of physicians whendiagnosing stroke or TIA, and significantly improve the speed at whichtherapy against the stroke or TIA can be administered.

The biomarkers also yield extensive evidence about the nature of thestroke or TIA and the type therapy which should be administered. Forexample, the respective levels of biomarkers can be evaluated todetermine whether the patient is suffering an ishemic or hemmorhagicstroke, or whether the patient is suffering from a traumatic braininjury. The data from the biomarkers can also be used to monitor orevaluate the progression of the ishemic episode, as well as the damagethat has resulted as a consequence of the ischemia. High levels of allparameters reflect the neurological deficit and may be also used forprognosis of disease outcome. Moreover, a relationship has been observedbetween the respective levels of the biomarkers and the degree ofthromboembolic and neurotoxicity in brain processes under the stroke.Once again, these relationships can be put to extensive use whenevaluating the choice of emergency therapy in short time frames, such asanti-platelet and neuroprotective therapy. The data can be usedindependently of other diagnostic strategies, but preferably forms anintegral part of a comprshensive diagnostic strategy employingconventional diagnostic techniques.

The data obtained from the NMDA biomarkers, especially when combinedwith data from other biomarkers such as glutamate and homocysteine, canalso be used to monitor the efficacy of a treatment regime. It hassurprisingly been found that the NMDA biomarkers provide real timeevidence of neurotoxicity, and that reductions in levels of circulatingNR1A or NR2A NMDA receptors or fragments thereof correspond well withreductions in neurotoxic mechanisms. By obtaining data at appropriateintervals using rapid laboratory techniques such as latex agglutination,one is able to monitor the progression of the episode in response to thetherapeutic regime.

A latex agglutination technique has also been developed whichdramatically increases the speed of diagnosis obtained by the methods ofthis invention, and thereby improves the effectiveness of the methods inemergency-room settings. The technique can be adapted for use in thedetection of NMDA receptors, homocysteine, glutamate, or any othersuitable biomarker against central nervous system disorders. Using thelatex agglutination technique, one is able to provide real-timebiochemical diagnosis and monitoring of TIA/stroke patients (withinabout 30 minutes), and thereby dramatically improve, the effectivenessof response to TIA/stroke. This is surprising because these biomarkersare naturally occurring and, in contrast to viruses for which latexagglutination methods were originally developed, show much lowerstrengths of association with their corresponding antibodies.

This semi-quantitative method gives reliable data quickly in a formatthat is simple for interpretation. Surprisingly, the technique showsgreater accuracy than even well established methods based upon HPLC andELISA. The application of the latex agglutination technique to theanalysis of brain biomarkers for stroke will decrease the cost ofanalysis, provide the opportunity to monitor real-time progress of atreatment procedure, and allow physicians to determine the efficacy ofmedication administered in the treatment of TIA or stroke.

The methods of the present invention also can be employed in anon-emergency setting, when evaluating the risk that an individual willsuffer a stroke or TIA. In addition, based upon results showing anincreased risk of suffering TIA or stroke, prevention therapy can beadministered, and the effectiveness of the therapy monitored using themethods of the present invention.

The invention also relates to indirect methods for measuring levels ofNR2A and NR2B NMDA receptor proteins or fragments thereof. Thus,analytical techniques can be used to evaluate indirect measures of NR2Aand NR2B NMDA receptor proteins or fragments thereof, such asautoantibodies specific for the proteins, or cDNA that encodes for theproteins.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein. Before the present methods andtechniques are disclosed and described, it is to be understood that thisinvention is not limited to specific analytical or synthetic methods assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

DEFINITIONS AND USE OF TERMS

As used in this specification and in the claims which follow, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “afragment” includes mixtures of fragments, reference to “an cDNAoligonucleotide” includes more than one oligonucleotide, and the like.

An analogue of a protein, peptide, or polypeptide means a protein,peptide, or polypeptide that contains one or more amino acidsubstitutions, deletions, additions, or rearrangements. For example, itis well known in the art of protein biochemistry that an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic (such as charge, hydrophobicity, and hydrophilicity) canoften be substituted for another amino acid without altering theactivity of the protein, particularly in regions of the protein that arenot directly associated with biological activity. Thus, an analogue ofan NMDA receptor or fragment thereof is useful in the present inventionif it includes amino acid substitutions, deletions, additions orrearrangements at sites such that antibodies raised against the analogueare still specific against the NMDA receptor or fragment.

Preferably, an NMDA analogue has at least 80%, 85%, 90%, or 95% aminoacid identity with naturally occurring NMDA. Amino acid identity isdefined by a analoguey comparison between the analogue and naturallyoccurring NMDA. The two amino acid sequences are aligned in such a waythat maximizes the number of amino acids in common along the length oftheir sequences; gaps in either or both sequences are permitted inmaking the alignment in order to maximize the number of common aminoacids. The percentage amino acid identity is the higher of the followingtwo numbers: (1) the number of amino acids that the two polypeptideshave in common with the alignment, divided by the number of amino acidsin the NMDA analogue or fragment thereof, multiplied by 100, or (2) thenumber of amino acids that the two polypeptides have in common with thealignment, divided by the number of amino acids in naturally occurringNMDA or fragment thereof, multiplied by 100.

NMDA derivatives, and derivatives of NMDA fragments, include naturallyoccurring NMDA and NMDA analogues and fragments thereof that arechemically or enzymatically derivatized at one or more constituent aminoacids, including side chain modifications, backbone modifications, andN- and C-terminal modifications, by for example acetylation,hydroxylation, methylation, amidation, phosphorylation or glycosylation.The term also includes NMDA salts such as zinc NMDA and ammonium NMDA.

A protein or peptide is measured “directly” in the sense that theprotein or peptide is itself measured in the biological sample, asopposed to some other indirect measure of the protein or peptide such asautoantibodies to the protein or peptide, or cDNA associated with theexpression of the protein or peptide.

The term “antibody” is intended to be synonymous with “immunoglobulin.”As used herein, the term “antibody” is meant to include both the nativeantibody, and biologically active derivatives of antibodies, such as,for example, Fab′, F(ab′)₂ or Fv as well as single-domain andsingle-chain antibodies. A biologically active derivative of an antibodyretains the ability to bind antigen.

General Discussion

The present disclosure describes diagnostic and therapeutic applicationsthat result from the realization that genetic or accidental increase ofNMDA receptors synthesis in the brain reflects a neurological ischemicdeficit, and may be used for early diagnoses of stroke or TIA. NMDAreceptors that are abnormally expressed in the brain are quicklymetabolized and, following penetration of the blood brain barrier, thesemetabolic destruction products enter the circulatory system. The immunesystem recognizes these peptides and protein fragments as foreignantigens and responds by generating autoantibodies to them.

In one aspect of the present invention, the correlation betweenincreased NMDA receptor synthesis, and the appearance of high levels ofthe receptors in blood sera of individuals during the initial stages ofcerebral ischemia, is used for diagnostic and therapeutic applications.Experiments in rats with focal ischemia have demonstrated that NR2A mRNAexpression in the cortex and hippocampus can be measured within twohours of the onset of the ischemic episode, and thus provide anopportunity for real time measurement of ischemic processes and damageresulting therefrom. At the same time, meaningful expression of NR2C andNR2D mRNA is not observed in brain structures that showed no changes inNR1 mRNA expression in rat ischemic brain. These changes in NR2-receptormRNA expression in the early stages of ischemia are observed prior tomorphological evidence of neuronal damage or appearance ofautoantibodies to them in blood serum specimens.

Thus, in one aspect the present invention provides a method fordiagnosing a central nervous system disorder comprising measuring thelevel of NR2A and/or NR2B NMDA receptor or fragment thereof in abiological sample. Elevated levels of NR2A and NR2B NMDA receptors arespecific to brain injury, and are expressed in ischemic brain tissue athigher rates than other NMDA receptors, and thus are uniquely suited forassessing ischemic brain episodes such as TIA or stroke. Baseline levelsfor determining whether the measured levels are elevated, and henceindicative of a central nervous system disorder, can be obtained frompopulation norms or, preferably, from a patient's own test history.

The biological sample tested for the receptor or fragment can be derivedfrom blood, urine, blood plasma, blood serum, cerebrospinal fluid,saliva, perspiration, or brain tissue. In a preferred embodiment, thebiological sample is a blood sample. In an even more preferredembodiment the biological sample is a blood sample diluted to a ratio offrom about 1:2 to about 1:32 (v:v).

Immunoassay techniques are generally preferred for measuring theproteins or peptides of the present invention, as discussed in greaterdetail herein, although other analytical techniques are also availableas known to those skilled in the art, such as HPLC. The amino acidsequences of the NR2A and NR2B subunits, and antigenic fragmentsthereof, are recited in SEQ ID NOS.1, 2, 3, 10, 11, and 12, and anyfragment of these sequences can be employed in methods for directlydetecting the receptors as long as sufficient antigenicity ismaintained. However, when using immunoassays it has been found that theantigenic determinants are concentrated in the N-terminal domain of theNR2A and/or NR2B NMDA receptor, and that antibodies raised against theN-terminal domains and fragments thereof should be employed for optimaltest results. The inventors have sequenced the amino acid chain of theN-terminal domains for these receptors, and set forth the sequences asSEQ ID NOS. 2 and 11, respectively, at the end of this document.

In a preferred embodiment, other biomarkers of central nervous systemdisorders are also measured to improve the accuracy of the diagnosis,and to provide further information about the nature, severity, orprogression of the disorder. Particularly useful markers are directlyimplicated in the NMDA receptor pathway, and include naturally occurringagonists and antagonists of the NMDA receptors. An exemplary antagonistis glycine. Exemplary agonists include glutamate, polyglutamate,aspartate, polyaspartate, homocysteine, and polyhomocysteine. Aparticularly preferred agonist for measuring the activity of thereceptors is glutamate or polyglutamate.

In another embodiment, thromboembolic biomarkers are measured to obtaina simultaneous reading of the likelihood for clotting in the brain.Exemplary thromboembolic biomarkers include homocysteine orpolyhomocysteine.

Titers of higher than 2.63 for combined levels of NR2A and NR2B,especially when combined with titers higher than 3.34 for glutamateand/or 2.23 for homocysteine, are remarkably predictive of theoccurrence of stroke and typically justify immediate therapeuticintervention for the TIA or stroke or risk of stroke. These titers canbe translated into absolute concentrations by reference to the exampleshereof.

The methods of the present invention are preferably performed bydirectly measuring the levels of NR2A and/or NR2B biomarkers in aselected biological sample, using immunoassay techniques employingantibodies raised against the biomarkers, or through quantitativetechniques such as HPLC. However, it is also possible to measure thepresence of the NR2A and/or NR2B biomarkers indirectly. This can be doneby directly measuring autoantibodies of the biomarkers, or by directlymeasuring the cDNA nucleic acid intermediates involved in expression ofthese biomarkers. If autoantibodies are measured, they are preferablymeasured using one or more antigenic fragments of the NR2A and/or NR2Breceptors as the target of the antibody, as opposed to a whole NR2Aand/or NR2B protein. Healthy persons generally have NR2A autoantibodiesin an amount of about 1.0-2.0 ng/ml. Healthy persons generally have NR2AcDNA levels of about 1.0-1.5 pg/ml.

Latex Agglutination and Other Diagnostic Techniques

A number of immunoassays can be employed in accordance with theprinciples of the present invention. Examples include radioimmunoassays,enzyme immunoassays (e.g. ELISA), immunofluorescence,immunoprecipitation, latex agglutination, hemagglutination, andhistochemical tests. A particularly preferred method, however, becauseof its speed and ease of use, is latex agglutination.

Latex agglutination assays have been described in Beltz, G. A. et al.,in Molecular Probes: Techniques and Medical Applications, A. Albertiniet al., eds., Raven Press, New York, 1989, incorporated herein byreference. In the latex agglutination assay, antibody raised against aparticular biomarker is immobilized on latex particles. A drop of thelatex particles is added to an appropriate dilution of the serum to betested and mixed by gentle rocking of the card. With samples lackingsufficient levels of the biomarkers, the latex particles remain insuspension and retain a smooth, milky appearance. However, if biomarkersreactive with the antibody are present, the latex particles clump intovisibly detectable aggregates.

An agglutination assay can also be used to detect biomarkers wherein thecorresponding antibody is immobilized on a suitable particle other thanlatex beads, for example, on gelatin, red blood cells, nylon, liposomes,gold particles, etc. The presence of antibodies in the assay causesagglutination, similar to that of a precipitation reaction, which canthen be detected by such techniques as nephelometry, turbidity, infraredspectrometry, visual inspection, colorimetry, and the like.

The term latex agglutination is employed generically herein to refer toany method based upon the formation of detectable agglutination, and isnot limited to the use of latex as the immunosorbent substrate. Whilepreferred substrates for the agglutination are latex based, such aspolystyrene and polypropylene, particularly polystyrene, otherwell-known substrates include beads formed from glass, paper, dextran,and nylon. The immobilized antibodies, may be covalently, ionically, orphysically bound to the solid-phase immunoadsorbent, by techniques suchas covalent bonding via an amide or ester linkage, ionic attraction, orby adsorption. Those skilled in the art will know many other suitablecarriers for binding antibodies, or will be able to ascertain such,using routine experimentation.

Thus, in one embodiment, the method of measuring the NR2A and/or NR2BNMDA receptor, fragment therof, or other biomarker is by latexagglutination comprising:

(i) contacting the biological sample with poly- or monoclonal antibodiesbound on an agglutinating carrier to target biomakers for a sufficienttime period and under conditions to promote agglutination; and

(ii) reading a signal generated from the agglutination; wherein theamount of signal detected correlates to the titer of biomarkers presentin the sample.

The reaction is preferably read macroscopically against a darkbackground for a sufficient time period. The method preferably yields aclinically useful reading within about 30 minutes or less.

It has been experimentally found that latex beads having a mean diameterof from about 0.25 to about 0.4 μm are particularly preferred in thepractice of this invention. The poly- or monoclonal antibodies arepreferably present in a ratio with the latex beads of about 1:1.

Latex beads having the foregoing characteristics can be preparedgenerally by adding antibodies to the target biomarker to a carriersolution that contains a 1% concentration (by weight) of latex beads,until the concentration of the antibodioes in the carrier solutionreaches about 2 mg/ml, and allowing the ingredients a sufficient time tocovalently link, typically about 1 hour, in the presence of a linkingagent such as glutaraldehyde.

Conventional methods can be used to prepare the antibodies. For example,by using a peptide of a NMDA protein, polyclonal antisera or monoclonalantibodies can be made using standard methods. A mammal, (e.g., a mouse,hamster, or rabbit) can be immunized with an immunogenic form of thepeptide (preferably the NR2A and/or NR2B receptor, an antigenicdeterminant of the NR2A and/or NR2B receptor, or an analogue orderivative thereof) which elicits an antibody response in the mammal.Techniques for conferring immunogenicity on a peptide includeconjugation to carriers or other techniques well known in the art. Forexample, the peptide can be administered in the presence of adjuvant.The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassayprocedures can be used with the immunogen as antigen to assess thelevels of antibodies. Following immunization, antisera can beadministered and, if desired, polyclonal antibodies isolated from thesera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art, (e.g., the hybridoma technique originally developed by Kohlerand Milstein (Nature 256, 495-497 (1975)) as well as other techniquessuch as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4, 72 (1983)), the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al. Monoclonal Antibodies in CancerTherapy (1985) Allen R. Bliss, Inc., pages 77-96), and screening ofcombinatorial antibody libraries (Huse et al., Science 246, 1275(1989)]. Hybridoma cells can be screened immunochemically for productionof antibodies specifically reactive with the peptide and the monoclonalantibodies can be isolated. Therefore, the invention also contemplateshybridoma cells secreting monoclonal antibodies with specificity forNR2A or NR2B NMDA proteins or fragments thereof as described herein.

When the NR2A and/or NR2B receptors are detected indirectly, bymeasuring the cDNA expression of the NR2A and/or NR2B receptors, themeasuring step in the present invention may be carried out bytraditional PCR assays such as cDNA hybridization, Northern blots, orSouthern blots. These methods can be carried out using oligonucleotidesencoding the polypeptide antigens of the invention. Therefore, in oneembodiment the methods are performed employing oligonucleotides thatencode the amino acid sequence of SEQ ID NO: 2, which is preferablyrepresented by nucleotides 371-1978 of SEQ ID NO: 6. More preferably,the nucleic acid construct comprises a oligonucleotide consisting of anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3,which is preferably represented by oligonucleotides 1790-1852 of SEQ IDNO: 7.

Thus, in one embodiment the methods of this invention include measuringan increase of NR2A and/or NR2B cDNA expression by contacting the totalDNA isolated from a biological sample with oligonucleotide primersattached to a solid phase, for a sufficient time period. In anotherpreferred embodiment, NR2A and/or NR2B cDNA expression is measured bycontacting an array of total DNA bound to a solid matrix with aready-to-use reagent mixture containing oligonucleotide primers for asufficient time period. Expressed NR2A cDNA is revealed by thecomplexation of the cDNA with an indicator reagent that comprises acounterpart oligonucleotide to the cDNA attached to a signal-generatingcompound. The signal-generating compound is preferably selected from thegroup consisting of horseradish peroxidase, alkaline phosphatase,urinase and non-enzyme reagents. The signal-generating compound is mostpreferably a non-enzyme reagent.

In a preferred embodiment, the solid phase is a polymer matrix. Morepreferably, the polymer matrix is polyacrylate, polystyrene, orpolypropylene. In one preferred embodiment the solid phase is amicroplate. In another preferred embodiment, the solid phase is anitrocellulose membrane or a charged nylon membrane.

As mentioned above, the methods of performing the present invention alsomay be performed by measuring the levels of autoantibodies specific forthe NR2A and/or NR2B subunits. These autoantibodies may be measured byany suitable immunoassay such as, for example, a radioimmunoassay, animmunofluorescence assay, an enzyme-linked immunosorbent assay (ELISA),an immunocytochemical assay, and immunoblotting. In a preferredembodiment, the antigen to which the anti-NR2A and/or NR2Bautoantibodies bind is a polypeptide or protein fragment of theN-terminal domain of the NR2A and/or NR2B receptor. More preferably, theantigen comprises a polypeptide or protein fragment of amino acid SEQ IDNO:2, 3, 4, 11, 12, or 13, or a suitable analogue or derivative thereof.

Thus, in yet another embodiment the methods of the present invention areperformed by measuring the levels of anti-NR2A and/or anti-NR2Bautoantibodies, by contacting a biological sample with a polypeptide orprotein fragment of the NR2A and/or NR2B receptor (preferably theN-terminal domain) (or an analogue or derivative thereof) attached to asolid phase, for a sufficient time period and under conditions to allowa complex to form between any NR2A and/or NR2B autoantibodies which maybe present in the sample and the polypeptide or protein fragment,contacting the complex with an indicator reagent comprising a secondaryantibody specific for the species of the mammal attached to asignal-generating compound (or for the polypeptide or protein fragment);and measuring the signal generated. The peptide can be obtained directlyfrom biological samples, by recombinant DNA techniques, or by directchemical synthesis. The signal-generating compound is preferablyselected from horseradish peroxidase, alkaline phosphatase, and urinase.More preferably, the signal-generating compound is horseradishperoxidase. Most preferably, the indicator reagent is rabbit anti-humanIgG attached to horseradish peroxidase. The amount of signal detected iscorrelated to the amount of anti-NR2A and/or NR2B autoantibodies presentin the biological sample.

In this method it is preferred that the solid phase be a polymer matrix.More preferably, the polymer matrix is selected from the groupconsisting of polyacrylate, polystyrene, and polypropylene. In onepreferred embodiment the solid phase is a microplate. In anotherpreferred embodiment, the solid phase is a nitrocellulose membrane or acharged nylon membrane.

The immunosorbent of the present invention for measuring levels ofautoantibody can be produced as follows. A fragment of the receptorprotein is fixed, preferably by covalent bond or an ionic bond, on asuitable carrier such as polystyrene or nitrocellulose. If the standardpolystyrene plate for immunological examinations is employed, it is frstsubjected to the nitration procedure, whereby free nitrogroups areformed on the plate surface, which are reduced to amino groups andactivated with glutaric dialdehyde serving as a linker. Next thethus-activated plate is incubated with about 2 to 50 nM of the targetpeptide for the purpose of chemically fixing the respective immunogenicfragment of the receptor protein for a time and at a temperaturesufficient to assure fixation (i.e. for about 16 hours at 4° C.).

The amount of protein below 2 nM affects adversely the reliability ofthe findings, whereas its amount exceeding 50 nM is inexpedient due toan increase in the nonspecific binding of autoantibodies with theimmunosorbents. The plate is then washed with an aqueous solution ofsodium boron hydride and an aqueous solution of sodium chloride,vacuum-dried, enclosed in a hermetically sealed package, and put understorage at 4° C.

It is also practicable to produce the immunosorbent by fixing therespective fragment of the receptor protein on nitrocellulose strips byvirtue of ionic interaction. The respective fragment of the receptorprotein isolated from the mammals' brain is applied to nitrocelluloseand incubated for 15 min at 37° C. Then nitrocellulose is washed with a0.5% solution of Tween-20, and the resultant immunosobent is dried atroom temperature and stored in dry place for one year period.

Emergency Room Diagnosis and Prognosis

As mentioned above, the methods of the present invention are especiallywell suited for use in emergency room settings. There are two reasonsfor this. First, the method is extremely useful in an emergency roomsetting because NR2A and NR2B NMDA receptor levels are elevated at avery early stage of ishemic insult, and thus provide a real timeindication of neurotoxic events. This is in contrast to autoantibodieswhich require that an immune response first be mounted by the insultedorganism.

The second reason the method is useful in an emergency room setting isthe speed and ease with which the latex agglutination procedure can beemployed. Using the latex agglutination processes described herein, oneis able to turn laboratory results around often in less than 30 or even20 minutes. Thus, using the methods of the present invention real-timedata can be obtained that permits a therapeutic response within thewindow for an effective response to stroke.

Therefore, in one embodiment the invention provides a method fordiagnosing the existence of a central nervous system disorder such asTIA or stroke, further comprising withdrawing the biological sample froma human subject, wherein the biological sample is withdrawn within threehours of the onset of symptoms of the central nervous system disorder.In still another embodiment of the invention, the amount of time elapsedbetween withdrawing the biological sample from the subject, anddetecting or measuring the presence or quantity of the NR2A and/or NR2BNMDA receptor, is less than about one hour, 45 minutes, or 30 minutes.

One of the principal adavntages of the present invention is the abilityto distinguish ischemic episodes such as stroke from other braininjuries such as traumatic brain injury. Thus, in another embodiment,the invention provides a method for diagnosing the existence of TIA orstroke further comprising evaluating from the level of NR2A and/or NR2BNMDA receptor whether the brain injury is a traumatic brain injury orstroke/TIA, and administering traimatic brain injuty or stroke/TIAtherapy as appropriate.

Another advantage of the methods of the present invention which isextremely useful in an emergency room setting, is the ability todetermine from the test data the type of stroke involved. In particular,if a stroke is suspected, the method will help diagnose whether thestroke is an ischemic or hemorrhagic insult. Thus, in another embodimentthe invention provides a method for diagnosing the existence of TIA orstroke further comprising, when the diagnosis confirms a stroke,evaluating from the level of NR2A and/or NR2B NMDA receptor whether thestroke is ischemic or hemmorhagic and administering ischemic orhemmorhagic stroke therapy as appropriate.

Another advantage of the present invention is the ability to evaluateinfarction volume and extent of neurotoxicity from NMDA expression dataNMDA receptor expression research in an animal model of middle carotidartery occlusion has been employed to demonstrate such correlation.Thus, in still another embodiment the invention provides a method fordiagnosing the existence of TIA or stroke further comprising, if TIAand/or stroke is confirmed, evaluating from the level of NR2A and/orNR2B NMDA receptor cranial infarct volume, and administering therapyappropriate to the infarct volume.

Moreover, one can periodically repeat the procedure, to providecontinuous monitoring of a patient's state as interventional therapy isadministered, to monitor the effectiveness of a particular therapeuticregime. In this embodiment, it is preferable for the mammal to beconcurrently undergoing treatment for the disorder. More preferably, thesamples are collected at intervals from about 20 min to about 1 month.Even more preferably, the interval is from about 20 min. to about 2hours. Most preferably the samples are collected at an interval of about30 minutes. Thus, in still another embodiment the invention provides amethod for diagnosing the progression of TIA or stroke furthercomprising detecting or measuring the presence or quantity of a NR2Aand/or NR2B NMDA receptor in a biological sample one or more additionaltimes, at a frequency of less than about 6 hours.

Primary Care Physician Setting

In another application the method is used in a clinical setting todetermine an individual's risk of stroke, or to monitor theeffectiveness of risk reduction therapies. As mentioned above, a numberof therapies can be employed to reduce the risk of stroke in anindividual. The use of antiplatelet agents, particularly aspirin, is astandard treatment for patients at risk for stroke. People with atrialfibrillation (irregular beating of the heart) may be prescribedanticoagulants. The most important treatable factors linked to TIAs andstroke are high blood pressure, cigarette smoking, heart disease,carotid artery disease, diabetes, and heavy use of alcohol. Medical helpis available to reduce and eliminate these factors. Lifestyle changessuch as eating a balanced diet, maintaining healthy weight, exercising,and enrolling in smoking and alcohol cessation programs can also reducethese factors. When these therapies are administered it is desirable todetermine the effectiveness of the therapy.

Therefore, in one embodiment the invention provides a method forevaluating an individual's risk for TIA or stroke comprising measuringlevels of NR2A and/or NR2B NMDA receptors or fragments thereof in abiological sample from the individual, and comparing the levels to abaseline level. In one embodiment the baseline levels are derived frompopulation averages. In another embodiment the baseline levels arederived from the individual's own medical history.

In another embodiment the method is performed more than once to monitorthe reduction or increase in risk for stroke or TIA, optionally inconjunction with the administration of risk reduction therapy. In oneembodiment the method is performed at a frequency of from about one weekto about six months. In another embodiment the method is performed at afrequency of from about one month to about three months.

In a particularly preferred embodiment other biomarkers are alsomeasured to assess the risk for stroke or TIA. Particularly preferredbiomarkers for risk of stroke or TIA are glutamate and homocysteine.

NOVEL KITS OF THE PRESENT INVENTION

In another embodiment the invention provides kits for diagnosing centralnervous system disorders such as TIA, stroke, and traumatic braininjury. NR2A and/or NR2B antibodies or antigens may be incorporated intoimmunoassay diagnostic kits depending upon whether autoantibodies orNMDA receptors are being measured. A first container may include acomposition comprising an antigen or antibody preparation. Both antibodyand antigen preparations should preferably be provided in a suitabletitrated form, with antigen concentrations and/or antibody titers givenfor easy reference in quantitative applications.

The kits may also include an immunodetection reagent or label for thedetection of specific immunoreaction between the provided antigen and/orantibody, as the case may be, and the diagnostic sample. Suitabledetection reagents are well known in the art as exemplified byradioactive, enzymatic or otherwise chromogenic ligands, which aretypically employed in association with the antigen and/or antibody, orin association with a second antibody having specificity for firstantibody. Thus, the reaction is detected or quantified by means ofdetecting or quantifying the label. Immunodetection reagents andprocesses suitable for application in connection with the novel methodsof the present invention are generally well known in the art.

The reagents may also include ancillary agents such as buffering agentsand protein stabilizing agents, e.g., polysaccharides and the like. Thediagnostic kit may further include where necessary agents for reducingbackground interference in a test, agents for increasing signal,apparatus for conducting a test, calibration curves and charts,standardization curves and charts, and the like.

In a more particular aspect the invention relates to a rapid multiplepanel containing antibodies to the thromboembolic and neurotoxicitybiomarkers glutamate, homocysteine and NMDA receptors that employs latexagglutination. Thus, in one embodiment the invention provides a kit fordiagnosing central nervous system disorders comprising: (1) anagglutinating immunosorbent for NR2A and/or NR2B NMDA receptors orfragments thereof, and (2) a control such as saline or a knownconcentration of NR2A and/or NR2B receptor or fragment thereof In a morepreferred embodiment the kit further comprises an agglutinatingimmunosorbent for another biomarker for TIA/stroke, such as an agonistor antagonist of NR2A and/or NR2B, a thromboembolic marker, or moreparticularly glutamate or polyglutamate, and/or an agglutinatinghomocysteine or polyhomocysteine. The agglutinating immunosorbent ispreferably of the type discussed in greater detail above.

In another embodiment the invention relates to a kit for detecting NR2Aand/or NR2B receptors or fragments thereof that does not employ latexagglutination. Thus, in another embodiment the invention provides a kitfor diagnosing central nervous system disorders comprising: (1) animmunosorbent for NR2A and/or NR2B NMDA receptors or fragments thereof,and (2) an indicator reagent comprising secondary antibodies attached toa signal generating compound. The secondary antibodies can be specificfor the receptor or fragment, or for the primary antibodies in theimmunosorbent. In a preferred embodiment the kits further comprise animmunosorbent for glutamate or polyglutamate, and/or an immunosorbentfor homocysteine or polyhomocysteine, and secondary antibodies againstthe glutamate and/or homocysteine, or to the primary antibodies on theimmunosorbents against the glutamate or homocysteine. The immunosorbentpreferably comprises anti-antibodies for the biomarkers bound to a solidsupport.

In another aspect the present invention relates to a test-kit thatrelies upon PCR amplification for measuring NR2A and/or NR2B levels.Thus, in another embodiment the invention provides a kit comprising: (a)one or more oligonucleotide primers (preferably of SEQ ID NO: 8)attached to a solid phase, (b) indicator reagent attached to asignal-generating, compound capable of generating a detectable signalfrom oligonucleotides, and (c) a control sample (i.e. template cDNA).The reagents may also include ancillary agents such as buffering agents,polymerase agents, and the like. The diagnostic kit may further include,where necessary, other members of the signal-producing system of whichsystem the detectable group is a member (e.g., enzyme and non-enzymesubstrates), agents for reducing background interference in a test,agents for increasing the signal, apparatus for conducting a test, andthe like.

In another embodiment of test-kit comprises (a) a solid phase to whichbiological fluids for receiving total DNA including NR2A cDNA could beattached, (b) oligonucleotide primers, preferably in a ready-to-use PCRbuffer, and (c) a control sample (i.e. template cDNA). Ancillary agentsas described above may similarly be included.

In another embodiment the invention provides a diagnostic kit fordetecting NR2A and/or NR2B autoantibodies comprising (a) a polypeptideof the N-terminal domain of the NR2A and/or NR2B receptor, fragmentthereof, or analog or derivative thereof, (b) an indicator reagentcomprising a secondary antibody specific for the autoantibody or thepolypeptide attached to a signal-generating compound; and (c) a controlsample, such as a known concentration of NR2A and/or NR2B polyclonalantibodies. The reagents may also include ancillary agents such asbuffering agents and protein stabilizing agents, e.g., polysaccharidesand the like. The diagnostic kit may further include, where necessary,other members of the signal-producing system of which system thedetectable group is a member (e.g., enzyme and non-enzyme substrates),agents for reducing background interference in a test, agents toincrease the signal, apparatus for conducting a test, calibration andstandardization information or instructions, and the like.

NOVEL COMPOSITIONS OF THE INVENTION

The methods of the present invention rely upon a series of novelcompositions which themselves form a part of the invention. Thus, in oneseries of embodiments the invention provides an isolated polypeptidefragment of the NR2A and/or NR2B NMDA receptor, comprising:

1. An antigenic determinant of the NR2A NMDA receptor,

2. An antigenic determinant of the NR2B NMDA receptor,

3. The N-terminal domain of the NR2A NMDA receptor,

4. The N-terminal domain of the NR2B NMDA receptor,

5. SEQ ID NO. 2,

6. SEQ ID NO. 3,

7. SEQ ID NO. 4,

8. SEQ ID NO. 11,

9. SEQ ID NO. 12, and

10. SEQ ID NO. 13,

or an antigenic fragment, analog, or derivative thereof. In anotherseries of embodiments the invention provides any, of the foregoingpolypeptides linked covalently to a distinct antigenic determinant, suchas human serum albumin. In still another series of embodiments theinvention provides any of the foregoing polypeptides linked to any ofthe immunosorbent materials discussed above. The immunosorbent can be inthe form of a bead for latex agglutination, in the size ranges discussedabove, or in the form of a synthetic plate for conventional immunoassayanalysis. The polypeptide can be linked to the immunosorbent using anyconventional means of linkage, including covalent linkage, ioniclinkage, and adsorption.

In another series of embodiments the present invention relates to thenovel monoclonal and polyclonal antibodies specific for and/or raisedagainst the foregoing polypeptides, including the foregoing polypeptideslinked to distinct antigenic determinants. Thus, in one embodiment theinvention provides non-human antibodies against any of the foregoingpeptides or polypeptides or antigenic fragment, analog, or derivativethereof. In another embodiment the invention provides immunosorbents towhich such antibodies are linked.

In another series of embodiments the present invention providesoligonucleotides that encode the foregoing peptides and polypeptides andfragments, analogs, and derivatives thereof, and to recombinantexpression vectors that include such oligonucleotides. Sucholigonucleotides include, without limitation, the oligonucleotidesdefined by SEQ ID NO:6, 7, 14, and 15, and fragments thereof whichencode antigenic determinants.

In still another embodiment the present invention relates to isolatedoligonucleotide sequences that are useful in the cDNA PCR analyticaltechniques of the present invention. Thus, the invention furtherprovides oligonucleotides comprising the nucleotide sequences of SEQ IDNOS:7, 8, 9, 15, 16, and 17.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

The features, aspects, and advantages of the present invention willbecome better understood with regard to the following sequence listingswhere, in the sequence the recited amino acid position numberingreflects that used throughout this document.

SEQ ID NO:1. shows the full-length amino acid sequence of the matureNR2A receptor subunit, as follows: SEQUENCE LISTING PEPTIDE Homo sapiensglutamate receptor. ionotropic, N-methyl D-aspartate 2A Science256:1217-1221(1992) NCBI/NM 000833.2 1         11         21         31         41         51 1 MGRLGYWTLLVLPALLVWRD PAQNAAAEKG PPALNIAVLL GHSHDVTERE LRNLWGPEQA 60 61 TGLPLDVNVVALLMNRTDPK SLITHVCDLM SGARIHGLVF GDDTDQEAVA QMLDFISSQT 120 121FIPILGIHGG ASMIMADKDP TSTFFQFGAS IQQQATVMLK IMQDYDWHVF SLVTTIFPGY 180181 RDFISFIKTT VDNSFVGWDM QNVITLDTSF EDAKTQVQLK KIHSSVILLY CSKDEAVLIL240 241 SEARSLGLTG YDFFWIVPSL VSGNTELIPK EFPSGLISVS YDDWDYSLEARVRDGLGILT 300 301 TAASSMLEKF SYIPEAKASC YGQAEKPETP LHTLHQFMVNVTWDGKDLSF TEEGYQVHPR 360 361 LVVIVLNKDR EWEKVGKWEN QTLSLRHAVWPRYKSFSDCE PDDNHLSIVT LEEAPFVIVE 420 421 DIDPLTETCV RNTVPCRKFVKINNSTNEGM NVKKCCKGFC IDILKKLSRT VKFTYDLYLV 480 481 TNGKHGKKVNNVWNGMIGEV VYQRAVMAVG SLTINEERSE VVDFSVPFVE TGISVMVSRS 540 541NGTVSPSAFL EPFSASVWVM MFVMLLIVSA IAVFVFEYFS PVGYNRNLAK GKAPHGPSFT 600601 IGKAIWLLWG LVFNNSVPVQ NPKGTTSKIM VSVWAFFAVI FLASYTANLA AFMIQEEFVD660 661 QVTGLSDKKF QRPHDYSPPF RFGTVPNGST ERNTRNNYPY MHQYMTRFNQRGVEDALVSL 720 721 KTGKLDAFIY DAAVLNYKAG RDEGCKLVTI GSGYIFASTGYGIALQKGSP WKRQIDLALL 780 781 QFVGDGEMEE LETLWLTGIC HNEKNEVMSSQLDIDNMAGV FYMLAAAMAL SLITFIWEHL 840 841 FYWKLRFCFT GVCSDRPGLLFSISRGIYSC IHGVHIEEKK KSPDFNLTGS QSNMLKLLRS 900 901 AKNISNMSNMNSSRMDSPKR ATDFIQRGSL IVDMVSDKGN LIYSDNRSFQ GKDSIFGDNM 960 961NELQTFVANR HKDNLSNYVF QGQHPLTLNE SNPNTVEVAV STESKGNSRP RQLWKKSMES 10201021 LRQDSLNQNP VSQRDEKTAE NRTHSLKSPR YLPEEVAHSD ISETSSRATC HREPDNNKNH1080 1081 KTKDNFKRSM ASKYPKDCSD VDRTYMKTKA SSPRDKIYTI DGEKRPSFHLDPPQFVENIT 1140 1141 LPENVGFPDT YQDHNENFRK GDSTLPMNRN PLHNEDGLPNNDQYKLYAKH FTLKDKGSPH 1200 1201 SEGSDRYRQN STHCRSCLSN LPTYSGHFTMRSPFKCDACL RMGNLYDIDE DQMLQETGNP 1260 1261 ATREEVYQQD WSQNNALQFQKNKLRINRQH SYDNILDKPR EIDLSRPSRS ISLKDRERLL 1320 1321 EGNLYGSLFSVPSSKLLGNK SSLFPQGLED SKRSKSLLPD HASDNPFLHT YGDDQRLVIG 1380 1381RCPSDPYKHS LPSQAVNDSY LRSSLRSTAS YCSRDSRGHS DVYISEHVMP YAANKNTMYS 14401441 TPRVLNSCSN RRVYKKMPSI ESDV

SEQ ID NO:2. shows the amino acid sequence of the auto-antigenic regionof the N-terminal domain of the NR2A subunit, as follows: SEQ ID NO:2HOMO SAPIENS PAQNAAAEKG PPALNIAVLL GHSHDVTERE LRNLWGPEQA 60 61TGLPLDVNVV ALLMNRTDPK SLITHVCDLM SGARIHGLVF GDDTDQEAVA QMLDFISSQT 120121 FIPILGIHGG ASMIMADKDP TSTFFQFGAS IQQQATVMLK IMQDYDWHVF SLVTTIFPGY180 181 RDFISFIKTT VDNSFVGWDM QNVITLDTSF EDAKTQVQLK KIHSSVILLYCSKDEAVLIL 240 241 SEARSLGLTG YDFFWIVPSL VSGNTELIPK EFPSGLISVSYDDWDYSLEA RVRDGLGILT 300 301 TAASSMLEKF SYIPEAKASG YGQAEKPETPLHTLHQFMVN VTWDGKDLSF TEEGYQVHPR 360 361 LVVIVLNKDR EWEKVGKWENQTLSLRHAVW PRYKSFSDCE PDDNHLSIVT LEEAPFVIVE 420 421 DIDPLTETCVRNTVPCRKFV KINNSTNEGM NVKKCCKGFC IDILKKLSRT VKFTYDLYLV 480 481TNGKHGKKVN NVWNGMIGEV VYQRAVMAVG SLTINEERSE VVDFSVPFVE TGISVMVSRS 540541 NGTVSPSAFL EPFSAS

SEQ ID NO:3; shows a 21 amino acid antigenic peptide, corresponding to afragment of the NR2A N-terminal domain.another such peptide (21 aminoacids derived from the NR2A sequence and an N-terminal Cys forattachment to a carrier protein), as follows: SEQ ID NO:3 Homo sapiensNGMIGEVVYQRAVMAVGSLTI

SEQ ID NO:4. shows a 22 amino acid antigenic peptide, corresponding to afragment of the NR2A N-terminal domain.another such peptide, modified byan N-terminal Cys for attachment to a carrier protein): ArtificialSequence CNGMIGEVVYQRAVMAVGSLTIFullBase Count OriginHomo sapiens glutamate receptor, ionotropic, N-methyl D-aspartate 2A(GRIN 2A) mRNA

SEQ ID NO:5. shows the Oligonucleotide position numbering usedthroughout in reference to NR2A oligonucleotide sequences, as follows:SEQ ID NO:5 Science 256:1217-1221(1992) May 22, 1992 NIGB/NM_000833 1atcatgggac cgggtgagcg ctgagaatcg cggccgcagc catcagccct ggagatgacc 61aggagcggcc actgctgaga actatgtgga gagaggctgc gagccctgct gcagagcctc 121cggctgggat agccgccccc cgtgggggcg atgcggacag cgcgggacag ccaggggagc 181gcgctggggc cgcagcatgc gggaacccgc taaacccggt ggctgctgag gcggccgaga 241tgctcgtgcg cgcagcgcgc cccactgcat cctcgacctt ctcgggctac agggaccgtc 301agtggcgact atgggcagag tgggctattg gaccctgctg gtgctgccgg cccttctggt 361ctggcgcggt ccggcgccga gcgcggcggc ggagaagggt ccccccgcgc taaatattgc 421ggtgatgctg ggtcacagcc acgacgtgac agagcgcgaa cttcgaacac tgtggggccc 481cgagcaggcg gcggggctgc ccctggacgt gaacgtggta gctctgctga tgaaccgcac 541cgaccccaag agcctcatca cgcacgtgtg cgacctcatg tccggggcac gcatccacgg 601cctcgtgttt ggggacgaca cggaccagga ggccgtagcc cagatgctgg attttatctc 661ctcccacacc ttcgtcccca tcttgggcat tcatgggggc gcatctatga tcatggctga 721caaggatccg acgtctacct tcttccagtt tggagcgtcc atccagcagc aagccacggt 781catgctgaag atcatgcagg attatgactg gcatgtcttc tccctggtga ccactatctt 841ccctggctac agggaattca tcagcttcgt caagaccaca gtggacaaca gctttgtggg 901ctgggacatg cagaatgtga tcacactgga cacttccttt gaggatgcaa agacacaagt 961ccagctgaag aagatccact cttctgtcat cttgctctac tgttccaaag acgaggctgt 1021tctcattctg agtgaggccc gctcccttgg cctcaccggg tatgatttct tctggattgt 1081ccccagcttg gtctctggga acacggagct catcccaaaa gagtttccat cgggactcat 1141ttctgtctcc tacgatgact gggactacag cctggaggcg agagtgaggg acggcattgg 1201catcctaacc accgctgcat cttctatgct ggagaagttc tcctacatcc ccgaggccaa 1261ggccagctgc tacgggcaga tggagaggcc agaggtcccg atgcacacct tgcacccatt 1321tatggtcaat gttacatggg atggcaaaga cttatccttc actgaggaag gctaccaggt 1381gcaccccagg ctggtggtga ttgtgctgaa caaagaccgg gaatgggaaa aggtgggcaa 1441gtgggagaac catacgctga gcctgaggca cgccgtgtgg cccaggtaca agtccttctc 1501cgactgtgag ccggatgaca accatctcag catcgtcacc ctggaggagg ccccattcgt 1561catcgtggaa gacatagacc ccctgaccga gacgtgtgtg aggaacaccg tgccatgtcg 1621gaagttcgtc aaaatcaaca attcaaccaa tgaggggatg aatgtgaaga aatgctgcaa 1681ggggttctgc attgatattc tgaagaagct ttccagaact gtgaagttta cttacgacct 1741ctatctggtg accaatggga agcatggcaa gaaagttaac aatgtgtgga atggaatgat 1801cggtgaagtg gtctatcaac gggcagtcat ggcagttggc tcgctcacca tcaatgagga 1861acgttctgaa gtggtggact tctctgtgcc ctttgtggaa acgggaatca gtgtcatggt 1921ttcaagaagt aatggcaccg tctcaccttc tgcttttcta gaaccattca gcgcctctgt 1981ctgggtgatg atgtttgtga tgctgctcat tgtttctgcc atagctgttt ttgtctttga 2041atacttcagc cctgttggat acaacagaaa cttagccaaa gggaaagcac cccatgggcc 2101ttcttttaca attggaaaag ctatatggct tctttggggc ctggtgttca ataactccgt 2161gcctgtccag aatcctaaag ggaccaccag caagatcatg gtatctgtat gggccttctt 2221cgctgtcata ttcctggcta gctacacagc caatctggct gccttcatga tccaagagga 2281atttgtggac caagtgaccg gcctcagtga caaaaagttt cagagacctc atgactattc 2341cccacctttt cgatttggga cagtgcctaa tggaagcacg gagagaaaca ttcggaataa 2401ctatccctac atgcatcagt acatgaccaa atttaatcag aaaggagtag aggacgcctt 2461ggtcagcctg aaaacgggga agctggacgc tttcatctac gatgccgcag tcttgaatta 2521caaggctggg agggatgaag gctgcaagct ggtgaccatc gggagtgggt acatctttgc 2581caccaccggt tatggaattg cccttcagaa aggctctcct tggaagaggc agatcgacct 2641ggccttgctt cagtttgtgg gtgatggtga gatggaggag ctggagaccc tgtggctcac 2701tgggatctgc cacaacgaga agaacgaggt gatgagcagc cagctggaca ttgacaacat 2761ggcgggcgta ttctacatgc tggctgccgc catggccctt agcctcatca ccttcatctg 2821ggagcacctc ttctactgga agctgcgctt ctgtttcacg ggcgtgtgct ccgaccggcc 2881tgggttgctc ttctccatca gcaggggcat ctacagctgc attcatggag tgcacattga 2941agaaaagaag aagtctccag acttcaatct gacgggatcc cagagcaaca tgttaaaact 3001cctccggtca gccaaaaaca tttccagcat gtccaacatg aactcctcaa gaatggactc 3061acccaaaaga gctgctgact tcatccaaag aggttccctc atcatggaca tggtttcaga 3121taaggggaat ttgatgtact cagacaacag gtcctttcag gggaaagaga gcatttttgg 3181agacaacatg aacgaactcc aaacatttgt ggccaaccgg cagaaggata acctcaataa 3241ctatgtattc cagggacaac atcctcttac tctcaatgag tccaacccta acacggtgga 3301ggtggccgtg agcacagaat ccaaagcgaa ctctagaccc cggcagctgt ggaagaaatc 3361cgtggattcc atacgccagg attcactatc ccagaatcca gtctcccaga gggatgaggc 3421aacagcagag aataggaccc actccctaaa gagccctagg tatcttccag aagagatggc 3481ccactctgac atttcagaaa cgtcaaatcg ggccacgtgc cacagggaac ctgacaacag 3541taagaaccac aaaaccaagg acaactttaa aaggtcagtg gcctccaaat accccaagga 3601ctgtagtgag gtcgagcgca cctacctgaa aaccaaatca agctccccta gagacaagat 3661ctacactata gatggtgaga aggagcctgg tttccactta gatccacccc agtttgttga 3721aaatgtgacc ctgcccgaga acgtggactt cccggacccc taccaggatc ccagtgaaaa 3781cttccgcaag ggggactcca cgctgccaat gaaccggaac cccttgcata atgaagaggg 3841gctttccaac aacgaccagt ataaactcta ctccaagcac ttcaccttga aagacaaggg 3901ttccccgcac agtgagacca gcgagcgata ccggcagaac tccacgcact gcagaagctg 3961cctttccaac atgcccacct attcaggcca cttcaccatg aggtccccct tcaagtgcga 4021tgcctgcctg cggatgggga acctctatga catcgatgaa gaccagatgc ttcaggagac 4081aggtaaccca gccaccgggg agcaggtcta ccagcaggac tgggcacaga acaatgccct 4141tcaattacaa aagaacaagc taaggattag ccgtcagcat tcctacgata acattgtcga 4201caaacctagg gagctagacc ttagcaggcc ctcccggagc ataagcctca aggacaggga 4261acggcttctg gagggaaatt tttacggcag cctgtttagt gtcccctcaa gcaaactctc 4321ggggaaaaaa agctcccttt tcccccaagg tctggaggac agcaagagga gcaagtctct 4381cttgccagac cacacctccg ataacccttt cctccactcc cacagggatg accaacgctt 4441ggttattggg agatgcccct cggaccctta caaacactcg ttgccatccc aggcggtgaa 4501tgacagctat cttcggtcgt ccttgaggtc aacggcatcg tactgttcca gggacagtcg 4561gggccacaat gatgtgtata tttcggagca tgttatgcct tatgctgcaa ataagaataa 4621tatgtactct acccccaggg ttttaaattc ctgcagcaat agacgcgtgt acaagaaaat 4681gcctagtatc gaatctgatg tttaaaaatc ttccattaat gttttatcta tagggaaata 4741cacgtaatgg ccaatgttct ggagggtaaa tgttggatgt ccaatagtgc cctgctaaga 4801ggaagaagat gtagggaggt attttgttgt tgttgttgtt ggctcttttg cacacggctt 4861catgccataa tcttccactc aaggaatctt gtgaggtgtg tgctgagcat ggcagacacc 4921agataggtga gtccttaacc aaaaataact aactacataa gggcaagtct ccgggacatg 4981cctactgggt atgttggcaa taatgatgca ttggatgcca atggtgatgt tatgatttcc 5041tatattccaa attccattaa ggtcagccca ccatgtaatt ttctcatcag aaatgcctaa 5101tggtttctct aatacagaat aagcaatatg gtgtgcatgt aaacctgaca cagacaaaat 5161aaaaacagtt aagaatgcat ctgcactgta gtcggatttg aacatgtgca agagattagg 5221aagtttggct cgtaacagtt tcagctttct tgttatgcct tccatcacag cccaggctca 5281ccccaagaac tccaggctcc cctaaagaat agcaaatcag tgtgttcgtg atgactgtgc 5341taccttcatt atagttcatt tccaagacac atctggagcc aaaggcccga gggaccctca 5401ggtggggaga gctacaggaa tctctttgga tgttgatgtg tgtttctctc taccctcggc 5461ttcgatggtc ttgttcagag ctgcataaac taacacattt atgtctccga gatctaagtg 5521tggatcttct gtctgtgaca cagtggccat tgtagtttat cccgaagacg cctatgtacg 5581taagtttgca tttcctccct tctggtgatg actcagggtt gtatagtatc tgttacccct 5641tccctcccag agtaaccata actcgttccg tttccaaaca gccatggtgg tgtccaatta 5701gctgtgtatc gctcttccca gagttgttaa tgtggtgaca tgcaccaaca gccgtatgtg 5761tactgtgatc tgtaagaagt acaatgccat ctgtctgccg aaggctagca tggttttagg 5821tttatcttcc ttcacatcca gaaattctgt tggacactca cttccacccc aaactcctca 5881aatcaaaagc cttcaaaaca cgaggcactc ttggatctac cctgagtatc ctccaaactg 5941tggatacagt ttagtgagac aagcaatttc tcccttctga gttattctct ctgttggtgg 6001caaaccactt catagcacca acagagatgt aggaaaaatt cctcaaagta tttgtcattt 6061ctgagtcgcc tgcattatcc cattcttatt ctcctcaaac ctgtgcatat atgacatgaa 6121atgatatcca tttttttttt aagttagaaa cagagagggg aatacttatg catggggagc 6181ctgttagcac agtgcctgcc acaaaaacaa gtgcccccga caagatagtt gctatgttat 6241gacactttct cagatcagga ttttctagtt taaaaattaa atatcataaa acg

SEQ ID NO:6. shows the oligonucleotide sequence of the auto-antigenicregion of the N-terminal domain of the NR2A subunit, as follows: SEQ IDNO:6 N-terminal nucleotide sequence 371 ccggcgccga gcgcggcggc ggagaagggtccccccgcgc taaatattgc 421 ggtgatgctg ggtcacagcc acgacgtgac agagcgcgaacttcgaacac tgtggggccc 481 cgagcaggcg gcggggctgc ccctggacgt gaacgtggtagctctgctga tgaaccgcac 541 cgaccccaag agcctcatca cgcacgtgtg cgacctcatgtccggggcac gcatccacgg 601 cctcgtgttt ggggacgaca cggaccagga ggccgtagcccagatgctgg attttatctc 661 ctcccacacc ttcgtcccca tcttgggcat tcatgggggcgcatctatga tcatggctga 721 caaggatccg acgtctacct tcttccagtt tggagcgtccatccagcagc aagccacggt 781 catgctgaag atcatgcagg attatgactg gcatgtcttctccctggtga ccactatctt 841 ccctggctac agggaattca tcagcttcgt caagaccacagtggacaaca gctttgtggg 901 ctgggacatg cagaatgtga tcacactgga cacttcctttgaggatgcaa agacacaagt 961 ccagctgaag aagatccact cttctgtcat cttgctctactgttccaaag acgaggctgt 1021 tctcattctg agtgaggccc gctcccttgg cctcaccgggtatgatttct tctggattgt 1081 ccccagcttg gtctctggga acacggagct catcccaaaagagtttccat cgggactcat 1141 ttctgtctcc tacgatgact gggactacag cctggaggcgagagtgaggg acggcattgg 1201 catcctaacc accgctgcat cttctatgct ggagaagttctcctacatcc ccgaggccaa 1261 ggccagctgc tacgggcaga tggagaggcc agaggtcccgatgcacacct tgcacccatt 1321 tatggtcaat gttacatggg atggcaaaga cttatccttcactgaggaag gctaccaggt 1381 gcaccccagg ctggtggtga ttgtgctgaa caaagaccgggaatgggaaa aggtgggcaa 1441 gtgggagaac catacgctga gcctgaggca cgccgtgtggcccaggtaca agtccttctc 1501 cgactgtgag ccggatgaca accatctcag catcgtcaccctggaggagg ccccattcgt 1561 catcgtggaa gacatagacc ccctgaccga gacgtgtgtgaggaacaccg tgccatgtcg 1621 gaagttcgtc aaaatcaaca attcaaccaa tgaggggatgaatgtgaaga aatgctgcaa 1681 ggggttctgc attgatattc tgaagaagct ttccagaactgtgaagttta cttacgacct 1741 ctatctggtg accaatggga agcatggcaa gaaagttaacaatgtgtgga atggaatgat 1801 cggtgaagtg gtctatcaac gggcagtcat ggcagttggctcgctcacca tc aatgagga 1861 acgttctgaa gtggtggact tctctgtgcc ctttgtggaaacgggaatca gtgtcatggt 1921 ttcaagaagt aatggcaccg tctcaccttc tgcttttctagaaccattca gcgcctct

SEQ ID NO:7 shows a 62 oligonucleotide fragment target, as follows: SEQID NO:7 atggaatgatcggtgaagtggtctatcaacgggcagtcatggcagttggc tcgctcaccatc

SEQ ID NO:8 shows one oligonucleotide primer, as follows: SEQ ID NO:8agcatggcaagaaagttaaca

SEQ ID NO:9 shows a second oligonucleotide primer, as follows: SEQ IDNO:9 acgttctgaagtggtggactt

SEQ ID NO: 10. shows the full-length amino acid sequence of the matureNR2B receptor subunit, as follows:

Peptide

Homo sapiens glutamate receptor, ionotropic, N-methyl D-aspartate 2B

Biochim. Biophys. Acta 1260:105-108(1995). sequence NME2_HUMAN (Q13224)1       11       21        31      41        51 1 MKPRAECCSP KFWLVLAVLAVSGSRARSQK SPPSIGIAVI LVGTSDEVAI KDAHEKDDFH 60 61HLSVVPRVEL VAMNETDPKS IITRICDLMS DRKIQGVVFA DDTDQEAIAQ ILDFISAQTL 120121 TPILGIHGGS SMIMADKDES SMFFQFGPSI EQQASVMLNI MEEYDWYIFS IVTTYFPGYQ180 181DFVNKIRSTI ENSFVGWELE EVLLLDMSLD DGDSKIQNQL KKLQSPIILL YCTKEEATYI 240241 FEVANSVGLT GYGYTWIVPS LVAGDTDTVP AEFPTGLISV SYDEWDYGLP ARVRDGIAII300 301TTAASDMLSE HSFIPEPKSS CYNTHEKRIY QSNMLNRYLI NVTFEGRNLS FSEDGYQMHP 360361 KLVIILLNKE RKWERVGKWK DKSLQMKYYV WPRMCPETEE QEDDHLSIVT LEEAPFVIVE420 421SVDPLSGTCM RNTVPCQKRI VTENKTDEEP GYIKKCCKGF CIDILKKISK SVKFTYDLYL 480481 VTNGKHGKKI NGTWNGMIGE VVMKRAYMAV GSLTINEERS EVVDFSVPFI ETGISVMVSR540 541 SNGTVSPSAF LEPFSADVWV MMFVMLLIVS AVAVFVFEYF SPVGYNRCLADGREPGGPSF 600 601 TIGKAIWLLW GLVFNNSVPV QNPKGTTSKI MVSVWAFFAVIFLASYTANL AAFMIQEEYV 660 661 DQVSGLSDKK FQRPNDFSPP FRFGTVPNGSTERNIRNNYA EMHAYMGKFN QRGVDDALLS 720 721 LKTGKLDAFI YDAAVLNYMAGRDEGCKLVT IGSGKVFAST GYGIAIQKDS GWKRQVDLAI 780 781 LQLFGDGEMEELEALWLTGI CHNEKNEVMS SQLDIDNMAG VFYMLGAAMA LSLITFICEH 840 841LFYWQFRHCF MGVCSGKPGM VFSISRGIYS CIHGVAIEER QSVMNSPTAT MNNTHSNILR 900901 LLRTAKNMAN LSGVNGSPQS ALDFIRRESS VYDISEHRRS FTHSDCKSYN NPPCEENLFS960 961 DYISEVERTF GNLQLKDSNV YQDHYHHHHR PHSIGSASSI DGLYDCDNPPFTTQSRSISK 1020 1021 KPLDIGLPSS KHSQLSDLYG KFSFKSDRYS GHDDLIRSDVSDISTHTVTY GNIEGNAAKR 1080 1081 RKQQYKDSLK KRPASAKSRR EFDEIELAYRRRPPRSPDHK RYFRDKBGLR DFYLDQFRTK 1140 1141 ENSPHWEHVD LTDIYKERSDDFKRDSVSGG GPCTNRSHIK HGTGDKHGVV SGVPAPWEKN 1200 1201 LTNVEWEDRSGGNFCRSCPS KLHNYSTTVT GQNSGRQACI RCEACKKAGN LYDISEDNSL 1260 1261QELDQPAAPV AVTSNASTTK YPQSPTNSKA QKKNRNKLRR QHSYDTFVDL QKEEAALAPR 13201321 SVSLKDKGRF MDGSPYAHMF EMSAGESTFA NNKSSVPTAG HHHHNNPGGG YMLSKSLYPD1380 1381 RVTQNPFIPT FGDDQCLLHG SKSYFFRQPT VAGASKARPD FRALVTNKPVVSALHGAVPA 1440 1441 RFQKDICIGN QSNPCVPNNK NPRAFNGSSN GHVYEKLSSI

SEQ ID NO: 11. shows the amino acid sequence of the auto-antigenicregion of the N-terminal domain of the NR2B subunit, as follows: SEQ IDNO:11 Homo sapiens RSQK SPPSIGIAVI LVGTSDEVAI KDAHEKDDFH 60 61HLSVVPRVEL VAMNETDPKS IITRICDLMS DRKIQGVVFA DDTDQEAIAQ ILDFISAQTL 120121 TPILGLHGGS SMIMADKDES SMFFQFGPSI EQQASVMLNI MEEYDWYIFS IVTTYFPGYQ180 181 DFVNKIRSTI ENSFVGWELE EVLLLDMSLD DGDSKIQNQL KKLQSPIILLYCTKEEATYI 240 241 FEVANSVGLT GYGYTWIVPS LVAGDTDTVP AEFPTGLISVSYDEWDYGLP ARVRDGIAII 300 301 TTAASDMLSE HSFIPEPKSS CYNTHEKRIYQSNMLNRYLI NVTFEGRNLS FSEDGYQMHP 360 361 KLVIILLNKE RKWERVGKWKDKSLQMKYYV WPRMCPETEE QEDDHLSIVT LEEAPFVIVE 420 421 SVDPLSGTCMRNTVPCQKRI VTENKTDEEP GYIKKCCKGF CIDILKKISK SVKFTYDLYL 480 481VTNGKHGKKI NGTWNGMIGE VVMKRAYMAV GSLTINEERS EVVDFSVPFI ETGISVMVSR 540541 SNGTVSPSAF LEPFSAD

SEQ ID NO:12; shows a 20 amino acid antigenic peptide fragment of theNR2B subunit, as follows: SEQ ID NO:12 Homo sapiens GYIKKCCKGFCIDILKKISK

SEQ ID NO:13 shows a 21 amino acid sequence of an antigenic fragment ofthe NR2B subunit modified by an N-terminal Cys for attachment to acarrier protein, as follows: SEQ ID NO:13 Artificial Sequence (21aminoacids) CGYIKKCCKGF CIDILKKISK.FullBase Count Origin

SEQ ID NO:14 shows the oligonucleotide position numbering usedthroughout in reference to NR2B oligonucleotide sequences, as follows:SEQ. NO. 14 Homo sapiens glutamate receptor, ionotropic, N-methylD-aspartate 2B mRNA 1 ttgaatttgc atctcttcaa gacacaagat taaaacaaaatttacgctaa attggatttt 61 aaattatctt ccgttcattt atccttcgtc tttcttatgtggatatgcaa gcgagaagaa 121 gggactggac attcccaaca tgctcactcc cttaatctgtccgtctagag gtttggcttc 181 tacaaaccaa gggagtcgac gagttgaaga tgaagcccagagcggagtgc tgttctccca 241 agttctggtt ggtgttggcc gtcctggccg tgtcaggcagcagagctcgt tctcagaaga 301 gcccccccag cattggcatt gctgtcatcc tcgtgggcacttccgacgag gtggccatca 361 aggatgccca cgagaaagat gatttccacc atctctccgtggtaccccgg gtggaactgg 421 tagccatgaa tgagaccgac ccaaagagca tcatcacccgcatctgtgat ctcatgtctg 481 accggaagat ccagggggtg gtgtttgctg atgacacagaccaggaagcc atcgcccaga 541 tcctcgattt catttcagca cagactctca ccccgatcctgggcatccac gggggctcct 601 ctatgataat ggcagataag gatgaatcct ccatgttcttccagtttggc ccatcaattg 661 aacagcaagc ttccgtaatg ctcaacatca tggaagaatatgactggtac atcttttcta 721 tcgtcaccac ctatttccct ggctaccagg actttgtaaacaagatccgc agcaccattg 781 agaatagctt tgtgggctgg gagctagagg aggtcctcctactggacatg tccctggacg 841 atggagattc taagatccag aatcagctca agaaacttcaaagccccatc attcttcttt 901 actgtaccaa ggaagaagcc acctacatct ttgaagtggccaactcagta gggctgactg 961 gctatggcta cacgtggatc gtgcccagtc tggtggcaggggatacagac acagtgcctg 1021 cggagttccc cactgggctc atctctgtat catatgatgaatgggactat ggcctccccg 1081 ccagagtgag agatggaatt gccataatca ccactgctgcttctgacatg ctgtctgagc 1141 acagcttcat ccctgagccc aaaagcagtt gttacaacacccacgagaag agaatctacc 1201 agtccaatat gctaaatagg tatctgatca atgtcacttttgaggggagg aatttgtcct 1261 tcagtgaaga tggctaccag atgcacccga aactggtgataattcttctg aacaaggaga 1321 ggaagtggga aagggtgggg aagtggaaag acaagtccctgcagatgaag tactatgtgt 1381 ggccccgaat gtgtccagag actgaagagc aggaggatgaccatctgagc attgtgaccc 1441 tggaggaggc accatttgtc attgtggaaa gtgtggaccctctgagtgga acctgcatga 1501 ggaacacagt cccctgccaa aaacgcatag tcactgagaataaaacagac gaggagccgg 1561gttacatcaa aaaatgctgc aaggggttct gtattgacat ccttaagaaa atttctaaat 1621ctgtgaagtt cacctatgac ctttacctgg ttaccaatgg caagcatggg aagaaaatca 1681atggaacctg gaatggtatg attggagagg tggtcatgaa gagggcctac atggcagtgg 1741gctcactcac catcaatgag gaacgatcgg aggtggtcga cttctctgtg cccttcatag 1801agacaggcat cagtgtcatg gtgtcacgca gcaatgggac tgtctcacct tctgccttct 1861tagagccatt cagcgctgac gtatgggtga tgatgtttgt gatgctgctc atcgtctcag 1921ccgtggctgt ctttgtcttt gagtacttca gccctgtggg ttataacagg tgcctcgctg 1981atggcagaga gcctggtgga ccctctttca ccatcggcaa agctatttgg ttgctctggg 2041gtctggtgtt taacaactcc gtacctgtgc agaacccaaa ggggaccacc tccaagatca 2101tggtgtcagt gtgggccttc tttgctgtca tcttcctggc cagctacact gccaacttag 2161ctgccttcat gatccaagag gaatatgtgg accaggtttc tggcctgagc gacaaaaagt 2221tccagagacc taatgacttc tcaccccctt tccgctttgg gaccgtgccc aacggcagca 2281cagagagaaa tattcgcaat aactatgcag aaatgcatgc ctacatggga aagttcaacc 2341agaggggtgt agatgatgca ttgctctccc tgaaaacagg gaaactggat gccttcatct 2401atgatgcagc agtgctgaac tatatggcag gcagagatga aggctgcaag ctggtgacca 2461ttggcagtgg gaaggtcttt gcttccactg gctatggcat tgccatccaa aaagattctg 2521ggtggaagcg ccaggtggac cttgctatcc tgcagctctt tggagatggg gagatggaag 2581aactggaagc tctctggctc actggcattt gtcacaatga gaagaatgag gtcatgagca 2641gccagctgga cattgacaac atggcagggg tcttctacat gttgggggcg gccatggctc 2701tcagcctcat caccttcatc tgcgaacacc ttttctattg gcagttccga cattgcttta 2761tgggtgtctg ttctggcaag cctggcatgg tcttctccat cagcagaggt atctacagct 2821gcatccatgg ggtggcgatc gaggagcgcc agtctgtaat gaactccccc accgcaacca 2881tgaacaacac acactccaac atcctgcgcc tgctgcgcac ggccaagaac atggctaacc 2941tgtctggtgt gaatggctca ccgcagagcg ccctggactt catccgacgg gagtcatccg 3001tctatgacat ctcagagcac cgccgcagct tcacgcattc tgactgcaaa tcctacaaca 3061acccgccctg tgaggagaac ctcttcagtg actacatcag tgaggtagag agaacgttcg 3121ggaacctgca gctgaaggac agcaacgtgt accaagatca ctaccaccat caccaccggc 3181cccatagtat tggcagtgcc agctccatcg atgggctcta cgactgtgac aacccaccct 3241tcaccaccca gtccaggtcc atcagcaaga agcccctgga catcggcctc ccctcctcca 3301agcacagcca gctcagtgac ctgtacggca aattctcctt caagagcgac cgctacagtg 3361gccacgacga cttgatccgc tccgatgtct ctgacatctc aacccacacc gtcacctatg 3421ggaacatcga gggcaatgcc gccaagaggc gtaagcagca atataaggac agcctgaaga 3481agcggcctgc ctcggccaag tcccgcaggg agtttgacga gatcgagctg gcctaccgtc 3541gccgaccgcc ccgctcccct gaccacaagc gctacttcag ggacaaggaa gggctacggg 3601acttctacct ggaccagttc cgaacaaagg agaactcacc ccactgggag cacgtagacc 3661tgaccgacat ctacaaggag cggagtgatg actttaagcg cgactccatc agcggaggag 3721ggccctgtac caacaggtct cacatcaagc acgggacggg cgacaaacac ggcgtggtca 3781gcggggtacc tgcaccttgg gagaagaacc tgaccaacgt ggagtgggag gaccggtccg 3841ggggcaactt ctgccgcagc tgtccctcca agctgcacaa ctactccacg acggtgacgg 3901gtcagaactc gggcaggcag gcgtgcatcc ggtgtgaggc ttgcaagaaa gcaggcaacc 3961tgtatgacat cagtgaggac aactccctgc aggaactgga ccagccggct gccccagtgg 4021cggtgacgtc aaacgcctcc accactaagt accctcagag cccgactaat tccaaggccc 4081agaagaagaa ccggaacaaa ctgcgccggc agcactccta cgacaccttc gtggacctgc 4141agaaggaaga agccgccctg gccccgcgca gcgtaagcct gaaagacaag ggccgattca 4201tggatgggag cccctacgcc cacatgtttg agatgtcagc tggcgagagc acctttgcca 4261acaacaagtc ctcagtgccc actgccggac atcaccacca caacaacccc ggcggcgggt 4321acatgctcag caagtcgctc taccctgacc gggtcacgca aaaccctttc atccccactt 4381ttggggacga ccagtgcttg ctccatggca gcaaatccta cttcttcagg cagcccacgg 4441tggcgggggc gtcgaaagcc aggccggact tccgggccct tgtcaccaac aagccggtgg 4501tctcggccct tcatggggcc gtgccagccc gtttccagaa ggacatctgt atagggaacc 4561agtccaaccc ctgtgtgcct aacaacaaaa accccagggc tttcaatggc tccagcaatg 4621ggcatgttta tgagaaactt tctagtattg agtctgatgt ctgagtgagg gaacagagag 4681gttaaggtgg gtacgggagg gtaaggctgt gggtcgcgtg atgcgcatgt cacggagggt 4741gacgggggtg aacttggttc ccatttgctc ctttcttgtt ttaatttatt tatgggatcc 4801tggagttctg gttcctactg ggggcaaccc tggtgaccag caccatctct cctccttttc 4861acagttctct ccttcttccc cccgctgtca gccattcctg ttcccatgag atgatgccat 4921gggccctctc agcaggggag ggtagagcgg agaaaggaag ggctgcatgc gggcttcctc 4981ctggtgtgga agagctcctt gatatcctct ttgagtgaag ctgggagaac caaaaagagg 5041ctatgtgagc acaaaggtag cttttcccaa actgatcttt tcatttaggt gaggaagcaa 5101aagcatctat gtgagaccat ttagcacact gcttgtgaaa ggaaagaggc tctggctaaa 5161ttcatgctgc ttagatgaca tctgtctagg aatcatgtgc caagcagagg ttgggaggcc 5221atttgtgttt atatataagc ccaaaaatgc ttgcttcaac cccatgagac tcgatagtgg 5281tggtgaacag aacccaaggt cattggtggc agagtggatt cttgaacaaa ctggaaagta 5341cgttatgata gtgtcccccg gtgccttggg gacaagagca ggtggattgt gcgtgcatgt 5401gtgttcatgc acacttgcac ccatgtgtag tcaggtgcct caagagaagg caaccttgac 5461tctttctatt gtttctttca atatccccaa gcagtgtgat tgtttggctt atatacagac 5521agagatggcc atgtattacc tgaattttgg ctgtgtctcc cttcatcctt ctggaataag 5581gagaatgaaa attcttgata aagaagattc tgtggtctaa acaaaaaaag gcggtgagca 5641atcctgcaag aacaaggtac ataaacaagt cctcagtggt tggcaattgt ttcaaccagt 5701ttgaaccaag aactttccag gaaggctaaa gggaaaccga attttcacag ccatgattct 5761tttgcccaca cttgggagca aaagattcta caaagctctt ttgagcattt agactctcga 5821ctggccaagg tttggggaag aacgaagcca cctttgaaga agtaaggagt cgtgtatggt 5881agggtaagtg agagaggggg atgtttccaa tgctttgatc ccttcttact taacctgaag 5941ctagacgagc aggcttcttc cccccaaaac tgattacaac tgctacagag cagacagtta 6001agagaaatga gcttgacctt taagagaaat gagctgcact ccatgagtgc agctctggag 6061gtacgaaaag aggggaagag acttggaaat gggagacggg ggcagagagg gaccctccac 6121cacctctttg ggcctggctc cctgggaatg tgacttgagc ccagagtgaa cactcttggt 6181agaagccctt ctaccttcct gcaacacctt gtttccctct cagattgtac cattgag

SEQ ID NO:15 shows a 60 oligonucleotide fragment target, as follows: SEQID NO:15 g gttacatcaa aaaatgctgc aaggggttct gtattgacat ccttaagaaaatttctaaa

SEQ ID NO:16 shows one oligonucleotide primers (21 nucleotides), asfollows: SEQ ID NO:16 tcactgagaa taaaacagac g

SEQ ID NO:17 shows one oligonucleotide primers (21 nucleotides), asfollows: SEQ ID NO:17 t cacctatgac ctttacctgg

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds claimed herein are made and evaluated, and are intended to bepurely exemplary of the invention and are not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.) but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in ° C. or is at room temperature, and pressure is at or nearatmospheric.

Example 1 Preparation of Polyclonal Antibodies (IgG) to Glutamate andHomocysteine

Glutamate (polyglutamate, 10 aminoacids) or homocysteine(polyhomocysteine, 10 aminoacids) alone will not generate antibodieswhen injected into an animal. Therefore, polyglutamate andpolyhomocysteine were conjugated with human serum albumin for theimmunization to obtain polyclonal antibodies. For glutaraldehydeconjugation, polyglutamate or homocysteine (10 mg) and 40 mg bovineserum albumin (BSA, Sigma, St. Louis, Mo.) were incubated for 2 hr atroom temperature in 4 ml of PBS containing 5% glutaraldehyde. Thereaction was stopped by adding glycine to a final concentration of 0.2M, and the conjugate was dialyzed against PBS.

Rabbits were given initial injections of 1 mg of conjugated glutamate(polyglutamate) or homocysteine (polyhomocysteine) in complete Freund'sand subsequent increased doses of injections (2 mg) in incompleteFreund's adjuvant at successive 2 week intervals. All injections weregiven subcutaneously. The immunization period lasted for 110 days.Antibodies (IgG) were affinity purified according to standard procedures(Warr, G. W., Purification of antibodies, In: Antibody as a Tool, Eds.,Marchalonis, J. J., and G. W. Warr, J. Wiley, UK, pp. 59-96 (1982)) andwere shown to be selective for glutamate or homocysteine by ELISA assay.

Example 2 Preparation of Polyclonal Antibodies (IgG) to NR2A ReceptorPeptide

Using computer analysis of the hydrophobicity profile of human NR2A andNR2B NMDA receptors to predict the antigenic determinants in the proteinstructure, we selected fragments corresponding to the N-terminalsequence of human NR2A and NR2B receptor peptides for synthesis. Thefragments corresponded to the N-terminal sequence of the NR2A and NR2Breceptors, represented by SEQ ID NO: 1 and SEQ ID NO: 2 for the NR2A andNR2B receptors, respectively. The peptide fragments were reproducedusing solid-phase synthesis, and had a purity ranging from 90% to 98%.The peptide sequences were verified by amino acid analysis after acidhydrolysis. A mixture of NR2A and NR2B peptides (1:1) was conjugatedwith human serum albumin for the immunization to obtain polyclonalantibodies. For glutaraldehyde conjugation, 10 mg of the mixture ofpeptides and 40 mg human serum albumin (Sigma, St. Louis, Mo.) wereincubated for 1.45 hr at room temperature in 4 ml of PBS containing 5%glutaraldehyde. The reaction was stopped by adding glycine to a finalconcentration of 0.2 M, and the conjugate was dialyzed against PBS.

Rabbit polyclonal antibodies were raised against the NR2A-B peptides.Rabbits were given initial injections of 1 mg of conjugated peptides incomplete Freund's adjuvant and subsequent injections (0.5 mg) inincomplete Freund's adjuvant at successive 2 week intervals. Antibodieswere affinity purified according to standard procedures (Warr, G. W.,Purification of antibodies, In: Antibody as a Tool, Eds., Marchalonis,J. J., and G. W. Warr, J. Wiley, UK, pp. 59-96 (1982)) and were shown tobe selective for NR2A and NR2B NMDA receptors using an ELISA assay.

Example 3 Preparation of Latex Beads Containing Biomarker Antibodies

Three different sensitized latex beads containing IgG against glutamate,homocysteine and NR2A-B receptor peptides were prepared using two typesof blue polystyrene latex beads (diameter, 0.25 and 0.4 μM; Sigma, St.Louis, Mo.) as follows. A 1% suspension of latex beads in 50 mM PBS (1ml, pH 7.0) was mixed with an equal volume of corresponding IgG (2mg/ml) and incubated on a shaker at room temperature for 2 hours. Themixture was then washed twice with PBS by centrifugation at 9,500 g for5 min. The pellet was suspended in PBS containing 1% BSA overnight at 4°C. After being washed twice with PBS, the sensitized latex beads wereresuspended in latex diluent (50 mM PBS with 1% BSA) at a concentrationof 0.4% and stored at 4° C. until used.

Preliminary experiments with latex agglutination (LA) alone wereperformed to identify problems and to select the most desirable latexparticle size. Two types of commercial latex beads were coated withantibodies at various concentrations. Tests were initially performedwith the corresponding aminoacid or NR2A and NR2B receptor peptides ascontrols. Particle size and IgG concentration were found to be theprimary factors affect the sensitivity of the test. The most desirableparticle size was found to be 0.25 μm (blue latex) because particles ofthis size agglutinated each aminoacid and peptide specifically. HigherIgG concentrations showed higher sensitivities. Using blue latex beadcoated with 2 mg of IgG per ml, agglutination could be observed within30 min.

Example 4 Latex Agglutination Analysis of Blood Serum Specimens

Blood samples (5 ml) were collected using standard venipuncture clinicalprotocol, from patients with TIA, stroke and brain injury (n=30) andexamined at the laboratory of CIS Biotech, Inc. in Atlanta (Ga., USA).None of the patients had been treated with anticoagulants, and serumsamples were obtained from the clotted blood. All specimens were free ofvisible lipids, white blood cells, platelets, fibrin, mucus or othercontaminants that could cause “false positive” reactions. Platelets,white blood cells, mucus and fibrin were removed by centrifugation.Lipids were removed by filtration.

Specimens to be tested within 72 hours after collection were stored at2-8° C. For longer storage periods, −20° C. or colder is recommended.

The semi-quantitative analysis of glutamate, homocysteine and NR2A-Breceptor peptides in the serum samples is basically a three stepprocess: serum sample dilution, reaction of latex beads with serumsamples, and product analysis.

In previous experiments serial dilutions of the serum samples from 1:4to 1:64 in saline containing 4% glycerol for better agglutination wereperformed. The highest dilution in which agglutination was observedcorresponded to the sample titer.

Two 25 μl aliquots of coated latex beads containing the correspondingIgG were layered on a double-concave slide (Fisher Sci., Norcross, Ga.),one with 25 μl of the serum sample in serial dilution to be tested andone with 25 μl of PBS as a negative control. After gentle mixing withvortex, agglutination was judged macroscopically against a darkbackground. A negative reaction corresponded to a homogeneous lactescentbackground with no agglutination; a positive reaction corresponded to aclearly visible agglutination against the black backround and weaklyvisible agglutination on a slightly lactescent background.

The highest dilution at which agglutination occurs gives the titer ofthe sample. To obtain the approximate titer in μg/ml we used thefollowing calculation:Titer μg/ml=A×Dwhere A is the test sensitivity, and D is the highest dilution at whichagglutination occurs.

Example 5 Description of Patients

Patients observed in trials (n=68) included 9 with pre-stroke, 9 withTIA (mean age 52.0±3.0), 31 with acute ischemic stroke (mean age54.7±1.4) and 11 with mild brain injury (mean age 53.0±4.4). Clinicalevaluation of patients by neuroimaging (CT, MRI, arteriography, Dopplerultrasonography, EEG), detailed physical and neurologic examination andlaboratory tests was performed. Patients with TIA were characterized bycontra lateral weakness, dysphasia, transient blurring of vision orblindness, abnormal pulsation of the common carotid arteries,microemboli confined to the ipsilateral retina. Untreated patients withpre-stroke demonstrated altered state of consciousness, severe headache,nausea and vomiting, visual disturbances, and focal neurologicaldeficit, with some patients experiencing seizures.

The N-Score rating scale reported in “MCA Infarction” (Orgogozo, 1986)was used for evaluating the neurologic deficit in patients with acutecerebral stroke. The total score of acute cerebral stroke clinicalmanifestation differentiated severe patients (n=9, 11-35 scores) frompatients with mild (n=12, 36-55 scores) and moderate patients (n=10,60-90 scores). Most patients with acute cerebral ischaemia (61.3%)suffered ischemia in the carotid artery of left hemisphere. Arterialhypertension and cerebral atherosclerosis etiologically corresponded inall patients.

The patients with ischemia were divided into groups based on thedifferences between TIA, pre-stroke and acute ischemic pathogenicmechanisms. The clinical diagnosis was established on the basis ofroutine observations which included detailed neurological examinationand neuroimaging. Groups of TIA (n=9) and pre-stroke patients withchronic cerebral blood insufficiency (n=9) were identified byneurophysiological investigations.

Example 6 Detection of Glutamate and Homocysteine in the Blood ofPatients

Glutamate and homocysteine content were measured by standard highperformance liquid chromatography (HPLC) according to methods described(Perry I. J., Refsum H., Morris R. W., Ebrahim S. B., Ueland P. M.,Shaper A. G. Lancet. 1995, 346:1395-1398; Yamamoto T., Rossi S., StiefelM., Doppenberg E., Zauner A., Bullock R., Marmarou A. Acta Neurochir.Suppl. 1999, 75: 17-19). The limits of the normal range were 165.0μmol/L for glutamate (Table 1) and 8.0 μmol/L for homocysteine (Table2). Elevated glutamate and homocysteine amounts were detected in theblood of patients with acute stroke. However, approximately 66% of thesepatients had additional risk factors indicative of atheroscleroticprocesses such as high cholesterol and LDL levels (Denisenko T. V.,Skuliabin D., Gromov I., Cherkas Yi., Iluchina A., Dambinova S. A.,1998. Vopr. Med. Khimii. 44, 584-590, in Russian).

Abnormal glutamate and homocysteine plasma concentrations were observedmore frequently in patients with TIA than in patients with acute stroke.The positive predictive efficiency of plasma glutamate for TIA patientswas 56%. The positive predictive efficiency of plasma homocysteine forTIA patients was 66%. Baseline concentrations for glutamate andhomocysteine are 160 umol/L and 10 umol/L, respectively. Routinetreatment for TIA was found to consistently decrease the glutamate andhomocysteine levels in the blood of patients (data not shown).

In patients with pre-stroke, slightly elevated levels homocysteine wereobserved; levels of glutamate were unchanged (Tables 1, 2). In patientswith traumatic brain injury (TBI), glutamate levels were observed thatwere nearly twice the glutamate levels in healthy individuals; levels ofhomocysteine were up to 57% higher. TABLE 1 Glutamate concentration inthe blood of patients detected by HPLC HPLC predictive value TotalGlutamate Negative Positive Group N μmol/L N % N % Healthy individuals28 165.0 ± 28.2 19 67.8 9 32.2 TIA 9 200.0 ± 11.7 4 44.4 5 55.6Pre-stroke 9 163.7 ± 10.4 5 55.6 4 44.4 Acute stroke 31 172.1 ± 20.6 1341.9 18 58.1 TBI 11 305.0 ± 28.8 4 36.4 7 63.6

We also compared homocysteine concentrations in the blood of patientswith TIA and pre-stroke to homocystein concentrations in the blood ofpatients who have had stroke onsets. We observed that homocysteinecontent in the blood of patients depended on stage of the stroke, butthat homocysteine concentration did not correlate with the severity ofthe cerebral ischemia A significant decrease in homocysteine levels inpatients with acute stroke was observed after emergency therapy (datanot shown).

Latex agglutination was also employed to detect TIA/stroke biomarkers inthe blood serum of patients. The titer of plasma glutamate determined bylatex agglutination was 3.34±0.25 in the group of healthy volunteers.Homocysteine and glutamate trends observed using HPLC were similarlyobserved for different groups of patients observed by using the LAtechnique (Tables 3, 4). Thus, increased levels of glutamate andhomocysteine were similarly observed in the blood of patients with TIAand acute stroke using LA.

With respect to predictive efficiency, however, LA showed a surprisingimprovement over HPLC. For example, the LA method improved the positivepredictive efficiency of patients with TIA and acute stroke on the basisof glutamate content to more than 63% (Tables 1, 3). The negativepredictive value for healthy patients was similarly improved when usingthe LA technique (Tables 3, 4). The predictive value of the LA techniquein the group of patients with TBI was identical to the predictive valueusing HPLC. TABLE 2 Homocysteine concentration in the blood of patientsdetected by HPLC HPLC predictive value Total Homocysteine NegativePositive Group N μmol/L N % N % Healthy individuals 28  8.0 ± 1.7 2071.4 8 28.6 TIA 9 10.8 ± 1.3 3 33.3 6 66.4 Pre-stroke 9  9.0 ± 1.2 444.4 5 55.6 Acute stroke 31 11.5 ± 1.1 11 35.5 20 64.5 TBI 11 12.6 ± 2.14 36.4 7 63.6

TABLE 3 Detection of glutamate in the blood of patients by latexagglutination LA predictive value Total Glutamate Negative PositiveGroup N Titer N % N % Healthy individuals 28 3.34 ± 0.25 22 78.6 6 21.4TIA 9 4.52 ± 0.38 3 33.3 6 66.4 Pre-stroke 9 3.57 ± 0.32 4 44.4 5 55.6Acute stroke 31 4.34 ± 0.47 11 35.5 20 64.5 TBI 11 5.12 ± 0.62 4 36.4 763.6

TABLE 4 Detection of homocysteine the blood of patients by latexagglutination LA predictive value Total Homocysteine Negative PositiveGroup N Titer N % N % Healthy individuals 28 2.23 ± 0.21 21 75.0 7 25.0TIA 9 3.95 ± 0.37 3 33.3 6 66.4 Pre-stroke 9 2.89 ± 0.12 4 44.4 5 55.6Acute stroke 31 4.01 ± 0.41 10 32.3 21 67.7 TBI 11 4.74 ± 0.38 4 36.4 763.6

Example 7 The Detection of NR2A-B in the Blood of Patients

The excessive activation and damage of NMDA receptors is the result ofglutamate, aspartate and homocysteine neurotoxicity. Autoantibodies tohave been detected in previous work in the blood of patients with TIAand pre-stroke, supporting our hypothesis that cerebral ischemia causesneuronal damage and the appearance of autoantibodies to NMDA receptorsubunits (Gusev E. I., Skvortsova V. I., Alekseev A. A., Izykenova G.A., Dambinova S. A. S.S Korsakov's J. Neurol. & Psych. 1996, 5:68-72;Dambinova S. A., Izykenova G. A. J. High Nervous Activity. 1997, 47:439-446).

The titer of NR2A-B receptor peptides in the blood of healthy volunteersdetermined by LA was 2.63±0.92. Using the LA technique, we observed anincrease in the test efficiency in the group of healthy persons up to89% (Table 5). We also observed an improvement in the sensitivity of theLA test over ELISA. For example, patients with pre-stroke had slightlyincreased levels of NR2A-B receptor peptides over healthy volunteerswhen tested by ELISA, but had nearly double the level of NR2A-B receptorpeptides when measured by LA (Table 5, 6). We detected high levels ofNR2A-B receptor peptides using both ELISA and LA in the blood ofpatients with TIA and acute stroke, and observed comparable levels ofpredictive efficiency for each test.

Patients with TIA received routine treatment to improve braincirculation. Upon receiving treatment, NR2A-B levels decreased to levelscorresponding to those observed for the healthy individuals as thepatient's state normalized. As mentioned earlier, glutamate andhomocysteine contents also decreased during treatment, but it neverreached the levels observed in healthy individuals. TABLE 5 Detection ofNR2A-B receptor peptides in the blood of patients by latex agglutinationLA predictive value Total NR2A-B Negative Positive Group N Titer N % N %Healthy individuals 28 2.63 ± 0.92 25 89.3 3 10.7 TIA 9 7.34 ± 0.43 222.2 7 77.8 Pre-stroke 9 4.21 ± 0.26 2 22.2 7 77.8 Acute stroke 31 5.20± 1.71 4 9.7 27 87.1 TBI 11 3.99 ± 0.44 2 18.8 9 81.8

Completely different profiles of NR2A-B were revealed in the blood ofpatients with acute ischemic stroke. In the blood of patients (n=8) withsevere cerebral ischaemia (30.4±3.2 Orgogozo scores) NR2A-B receptorpeptides titer was 4 times higher than that for control group of healthyindividuals. The peptides titer for patients with mild to moderateischemic stroke (n=22, 49-62 Orgogozo scores) was slightly elevated incomparison with those with TIA. The tendency of slight decreases inNR2A-B receptor peptide levels was observed to the end of 30 days ofpatients' routine treatment, correlating with improvement in theneurological state. TABLE 6 Detection of NR2A-B receptor peptides in theblood of patients by ELISA ELISA assay results Total NR2A-B NegativePositive Group N Ng/ml N % N % Healthy individuals 28 18.2 ± 2.1 20 71.48 28.6 TIA 9 66.6 ± 4.1 2 22.2 7 77.8 Pre-stroke 9 23.7 ± 1.9 3 33.3 666.7 Acute stroke 31 73.4 ± 6.5 5 16.1 26 83.9 TBI 11 54.3 ± 4.9 3 27.38 72.7

It is necessary notice that efficiency of both laboratory assays todetect the NR2A-B receptor peptides in the blood of patients withTIA/stroke and traumatic brain injury have been determined as 78 and 82%correspondingly.

The simultaneous detection of all brain damage biomarkers: glutamate,homocysteine and NR2A-B receptor peptides in the blood patients by latexagglutination allowed to diagnose rapidly TIA/stroke with efficiency upto 85-89%. The simultaneously increased levels of all biomarkers in theblood reflect the neurological deficit and may be used also forprognosis of diseases outcome. The relation between these biomarkers isshowing the degree of thromboembolic and neurotoxicity, involvement inbrain processes underlying the ischemia. That fact is very important forchoosing the strategy of emergency therapy in short time.

Using the latex agglutination technique allowed us significantly cut offthe time of blood analysis from 3-8 hours in ELISA or HPLC to 30 min inLA. This RMP semi-quantitative test demonstrated the fast, simple forinterpretation and reliable data.

Example 8 Identification of cDNA Sequence Encoding AntigenicDeterminants of NMDA Receptors

It was necessary to first determine the cDNA sequence coding theimmunological fragment of NMDA receptors responsible for the appearanceof autoantibodies appearance. To find the most active peptide fragmentof NMDA receptors a standard molecular biology procedure was used.Immunopositive phage GT11 containing cDNA coding NMDA receptors wasisolated from a human cDNA library using autoantibodies to NMDAreceptors isolated from blood samples of patients with severe cerebralischemia or polyclonal antibodies to the NR2A receptor. An E. Colibacterial system was employed to express the phage GT11 cDNA (600 bp).The expression product was transferred to a MBmp11 vector and arestriction map was constructed by use of a standard restrictases' kit.Three unique sites of the cDNA fragment (PstI, BamHI, and PsaI) wererevealed, and the 5′-3′ oligonucleotide sequence orientation using KpnI,BamHI and EcoR1 was deduced. The oligonucleotide (target cDNA) obtainedwas sequenced and compared to the sequence of the NR2A glutamatereceptor (SEQ ID NO: 5) from the NCBI library. The target cDNAcorresponded to the N-ternimal domain of the NR2A receptor (620 bp) ofSEQ ID NO: 6, namely SEQ ID NO: 7. Primers for this target nucleotidewere designed. All the oligonucleotides were prepared by thephosphoramidite method on an Applied Biosystem 394 synthesizer and werepurified by reverse-phase high-pressure liquid chromatography (HPLC).The oligonucleotides used for detection and capture were synthesizedwith an amine arm at the 5′ end.

Example 9 PCR Analysis of Blood Serum Specimens

Blood samples (5 ml collected by venipuncture) from patients with TIAand pre-stroke (n=30) were collected according to standard clinicalprotocol and examined at the Department of Neurology of Human BrainInstitute, St. Petersburg Russia. The blood specimens were used fortotal DNA isolation or applied on FTA paper circles.

The quantitative analysis of NR2A cDNA expression in the serum samplesis basically a three step process: Total DNA isolation and purificationfrom sera of individuals; specific cDNA coding NR2A receptoramplification; and product analysis.

The total DNA isolated by DNAzol (Mol. Res. Center, Inc., Cincinnati,Ohio) or bound on FTA blood staining collection cards (LifeTechnologies, Inc., Gathersburg, Md.) serves as a template for thepolymerase chain reaction (PCR). In the first variant, the PCR assayuses a set of specially designed primers (50 pmol), immobilized on solidmatrix of microplates and amplifies a specific cDNA sequence (620 bp)coding the NR2A glutamate receptor. In a second variant, the PCR assayuses a master ready-to-use buffer and amplifies cDNA bond on FTA paper.Following amplification, the quantity of a product is determined byenzyme or non-enzyme color reaction with a substrate.

Using the DNAzol reagent for DNA isolation, the whole blood of eachindividual (0.5 ml) was combined with 1 ml DNAzol (Mol. Res. Center,Inc., Cincinnati, Ohio) for 5 min at room temperature and lysed (MackeyK. et al. Mol. Biotechnol. 9: 1-5 (1997). The organic phase (0.4 ml) ofeach sample was transferred to a clean tube and 0.4 ml isopropanol wasadded. The mixture was incubated for 5 min at room temperature andcentrifugated at 6,000 g for 6 minutes. The pellet was washed in 0.5 mlDNAzol and centrifugated at the same conditions. The total DNA pelletwas mixed with 1 ml of 75% ethanol and centrifuged at 6,000 g for 5minutes. Then the DNA pellet was diluted in 200 l of 8 mM NaOH andincubated at room temperature for 5 min followed by vortexing. AlkalineDNA solution was then neutralized with 160 l of 0.1 M HEPES, pH 7.4.

Immobilization of oligonucleotide probes (primers, SEQ ID NO:8) wasperformed as follows. A total of 100 l of 3× PBS buffer containing theprimers (150 nM) was dropped into each well of a 96-well microtiterplate (Fisher Sci., Suwanee, Ga.). After incubation for 2 h at 37° C. orovernight at room temperature, the plate was washed three times with 1×PBS buffer containing 0.05% (w/vol) Tween 20. The oligonucleotide-coatedplates were stable for 2 months at 4° C.

Direct PCR reactions were performed in a final volume of 50 l (Sisk R B.in book: Molecular diagnostics: for the clinical laboratorian. Ed. byColeman W B., and Tsongalis G J. Humana Press Inc., Totowa, N.J. 1997,pp.103-121). The total DNA (5 l), isolated from blood samples ofindividuals, to oligonucleotide-coated plate in dublicates and 45 l ofmaster ready-to-use buffer containing 1 l TaKaRa Z-Taq DNA polymerase(TaKaRa Biomedicals, Otsu, Shiga, Japan) 10 l AMV/Tfl 5× reactionbuffer, 1 l dNTP mix (Promega, Madison, Wis.) 2 l of 25 mM MgSO₄ wereadded and sealed. The 30-thermal cycles (98° C.—5 s, 66° C.—2 sec )amplification using programmable Gene Cycler thermocycler (Bio-Rad Lab.,Hempstead, UK) for 20 minutes was performed. Then 50 l of PicoGreenreagent (Mol. Probes, Inc., Eugene, Oreg.) were added to each PCRproducts and mixed on a shaker (BioTechniques 20:676 (1996). Sampleswere incubated 5 min at room temperature, protected from light. Afterincubation the fluorescence of the samples was measured using afluorescence microplate reader (Mol. Device, Sunnyvale, Calif.) andstandard fluorescein wavelengths (excitation 480 nm, emission 520 nm).The fluorescence value of the reagent blank was subtracted from that ofeach of the samples, and the data was employed to generate five-pointstandardization curves of fluorescence versus DNA concentration, from 25pg/ml to 25 ng/ml reaction of control target cDNA (50 ng/ml stock) withthe same Pico Green reagent.

The other method of total DNA isolation is follows. Whole blood wasspotted onto FTA paper and lysed, and samples of DNA immobilized withinthe matrix of the stain card were punched into a 3 mm (⅛″) diameterpaper (1 mm or 2 mm Harris Micro-Punch™) and amplified directly by theamplification mix (Mackey K. et al. Mol. Biotechnol. 9: 1-5 (1997).

The FTA Bloodstain Card is divided into 4 circles for at least 4different 120 l samples of EDTA collected whole blood. Samples of bloodwere dried at room temperature for at least 1 hour. A circle was drawnwith a #2 pencil around each blood to visualize where the blood had beenspotted after the FTA paper processing. The FTA Bloodstain Card was thenplaced in a small plastic tray and 50 ml of FTA Purification Reagent wasadded and incubated on a shaker for 5 minutes. FTA Purification Reagentwas replace 3 times with 25-50 ml of the fresh solution and shaken foran additional 5 minutes. Then 25-50 ml of TE-4 (10 mM Tris-HCl pH 8.0;0.1 mM EDTA pH 8.0) was added and the mixture incubated twice on ashaker for 5 minutes. The FTA Bloodstain Card was allowed to air drycompletely during 2 hours at room temperature. The samples were thenpunched from the cards using a 3 mm diameter punch or the HarrisMicro-Punch (1.2 mm or 2.0 mm), and transferred into correspondingmicroplate wells. PCR was then performed using the above-describedprocedure using regular PCR microplates and a ready-to-use buffercontaining primers.

Patients (n=30, the age of 44-77) were divided into two groups. Thefirst group of patients (n=12) were diagnosed with TIA in the carotidcirculatory system, according to the following neurological criteria.Neural dysfunction was localized to a specific vascular distribution;the duration of the attack was usually less that 15 minutes and neverexceeded 24 hours; and the patients did not have abnormal neurologicsigns between attacks. The second pre-stroke group (n=18) were diagnosedwith TIA in the vertebral-basilar circulatory system. The second groupof patients was subdivided on the basis of compensation ornon-compensation of neurological deficit. The third group (n=12)included patients with migraine and epilepsy.

The control group of healthy individuals (n=20) showed a level of NR2AcDNA expression of 1.2 0.11 pg/ml. The first group demonstrated slightlyelevated levels of NR2A cDNA expression of 1.7 0.13 pg/ml. The patientswith compensation of neurological deficit from the second group showed alevel of NR2A cDNA expression of 1.8 1.4 pg/ml. At the same time, thepatients without compensation of neurological deficit that possessedmore severe symptoms of TIA showed levels of NR2A cDNA expression of 3times the levels seen in healthy individuals. Patients sufferingmigraine and epilepsy did not show any increase of NR2A cDNA expressionwhen compared with the control group.

Example 10 Immunological Analysis of Blood Serum Specimens

Blood samples (10 ml, collected by venipuncture) from patients withcerebral ischemia (n=70), and healthy individuals (n=200), collectedaccording to standard clinical protocols, were examined at the NeurologyHospital of Russian Medical Academy (Moscow, Russia). The bloodspecimens were centrifugated (4000 g, 5 min, +4° C.) and the collectedserum stored at −70° C. for further analysis.

Computer analysis was employed to predict the antigenic determinants inthe NR2A receptor protein structure based on hydrophobicity profile(Hopp, T. P. and K. R. Woods, Proc. Natl. Acad. Sci. USA 6: 3824-3828(1981)) and antigenicity (Welling, G. W., et al., FEBS Lett. 188:215-218 (1985)). Based upon this analysis, the N-terminal sequence ofthe NR2A NMDA receptor was synthesized. This synthetic peptide, whichcorresponded to amino acid sequence (494-514) (Grandy, D K., et al.,Proc. Natl. Acad. Sci. U.S.A. 86: 9762-9766(1989) (SEQ ID NO:3) of humanNR2A NMDA, was produced by solid-phase synthesis in a NPS-400semi-automated synthesizer (Neosystem Lab, France) on MBHA resin usingthe BOC/Bzl strategy for the first two amino acids. The peptides werepurified by preparative HPLC on a DELTAPAC™C18 column (WatersChromatography, Milford, Mass.) in a H₂O/acetonitrile/0.015 TFA system.The purity of the peptides was determined by analytical HPLC and rangedfrom 90% to 98%. The peptide sequence was verified by amino acidanalysis after acid hydrolysis. This peptide was used in immunoassays ofblood serum from patients and healthy individuals.

A quantitative analysis of the level of NR2A autoantibodies in serumsamples was performed by enzyme-linked immunosorbent assay (ELISA) (Ngo,T. T. and H. M. Lenhoff, FEBS Lett. 116: 285-288 (1980)). The dilutedblood sera (1:50) and polyclonal antibodies to the NR2A peptide as astandard (0.01 ng/ml-400 ng/ml) were applied to the immunosorbent. Theplate was incubated for 1 h at 25° C. and then washed by 0.05 Mphosphate buffer, pH 7.4, containing 0.05% of TWEEN-20™. Rabbitantibodies to the human immunoglobulin labeled with horseradishperoxidase were added (Sigma, St. Louis, Mo.; 1:1000), and the plate wasincubated for 1 h at 25° C. After incubation the wells were washed twicein the same buffer. The reaction was revealed by o-phenylenediamine in0.05 M citrate buffer, pH 4.3 monitored at 490 nm on a microplate reader(BioRad, UK). The titer of NR2A autoantibodies in blood serum wasdetermined by ELISA using a standard curve of the absorbence units ofNR2A autoantibodies versus their concentration in a microtiter wellplate.

The synthetic peptide corresponding to the NR2A NMDA glutamate receptors(3 μg) were immobilized on a nitrocellulose membrane (0.45 μm,Shleicher-Shuell, Germany) in phosphate-buffered saline (PBS), pH 7.4,then washed 2-3 times in the same buffer. Membranes with immobilizedpeptide were incubated with the diluted serum (1:50) of cerebralischemia patients and other subjects for 1 h at 25° C., and then rinsed4 times with the PBS buffer. Secondary rabbit anti-human immunoglobulinsconjugated with horseradish peroxidase (Sigma, St. Louis, Mo.; 1:1000)were incubated with the membrane for 1 h at 25° C., then washed 4 timeswith PBS. The development of brown color was registered and thenquantitated by densitometry.

To provide a positive control or standard, rabbit polyclonal antibodieswere raised against NR2A synthetic peptide corresponding to amino acidsequence predicted from the cloned human NR2A protein (Science 256:1217-1221 (1992); SEQ ID NO:1). For glutaraldehyde conjugation, 10 mg ofpeptide and 40 mg of human serum albumin (Sigma, St. Louis, Mo.) wereincubated for 1.45 h at room temperature in 4 ml of PBS containing 5%glutaraldehyde. The reaction was stopped by adding glycine to a finalconcentration of 0.2 M, and the conjugate was dialyzed against PBS.Rabbits were given initial injections of 1 mg of conjugated peptide incomplete Freund's and subsequent injections of 0.5 mg of peptide inincomplete Freund's adjuvant at successive 2 week intervals. Antibodieswere affinity purified according standard procedure (Warr, G. W.,Purification of antibodies, In: Antibody as a Tool, Eds., Marchalonis,J. J., and G. W. Warr, J. Wiley, UK, pp. 59-96 (1982)) and were shown tobe selective for the NR2A NMDA glutamate receptor using Western blotanalysis.

The patients (men, n=30; women, n=40; age of 40-75) were admitted in thehospital within no more than six hours after the onset of an ishemicepisode. All patients were divided into three groups according to theseverity of the stroke: The first group had moderate ischemic stroke(n=25), manifested by moderate focal deficit (>60—Orgogozo scale). Thesecond group had severe stroke (n=30), manifested by mild disorders ofconsciousness, severe headache, meningeal sings, and pronounced focaldeficit (30-60—Orgogozo scale). The third group had extremely severestroke (n=15), accompanied by stupor-coma, signs of brain edema,autonomic dysfunction, and severe focal deficit (<30—Orgogozo scale).

The level of NR2A autoantibodies was measured in the blood serum ofhealthy persons (n≦200, age 35-75) as a control, and ranged from 0.3-1.5ng/ml. The NR2A autoantibody level in the 55 patients of the first andsecond groups was significantly greater than that in the control group(p<0.01). Levels of NR2A autoantibodies were monitored every three hoursduring the first day, and then up to 5th day after stroke. The level ofNR2A autoantibodies in the blood serum of patients with severe strokewas significantly higher than that in the blood serum of patients withmoderate stroke, especially in the 9-12 hours after the onset of astroke (p<0,05). The tendency for NR2A autoantibodies level to decreaseto the control level on the first day of stroke was registered in groupof patients with good neurological recovery (90,50,5 units on Orgogozoscale). It can be concluded that the dynamic changes in NR2Aautoantibodies level may predict a recovery period of patients afterischemic stroke.

Example 11 SPRIA Assay of Autoantibodies

The solid-phase radioimmunoassay (SPRIA) of autoantibodies is performedas follows: a 10% acetic acid solution is added for one minute to theCooker microtiter microplates (available from Dynatech Co., USA) foractivation, whereupon 0.1 ml of the blood serum under analysis (diluted1:40) is applied to the microplates and subjected to incubation for fourhours at 25° C. Then the microplate are washed with a 0.14 M sodiumchloride solution and 0.1 ml of a mixture of the respective fragment ofthe mammal's brain protein labeled by 125I in the presence ofnonlabelled one. The plates are incubated for 20 hours at 4° C. Oncompletion of incubation, the microplates are washed with a 0.14M sodiumchloride solution, after which each of the wells of the microplates iscut off and placed in gamma-counting vials.

Example 12 ELISA Assay of Autoantibodies

The enzyme-linked immunosorbent assay (ELISA) of autoantibodies iscarried out as follows: the samples of the blood serum diluted 1:40 or1:50 are applied to the respective immunosorbent. Then the platecarrying the immunosorbent is incubated for 30 min at 37° C., whereuponthe wells of the plate are washed with a 0.05 M phosphate buffer,containing 0.05% of Tween-20. Rabbit antibodies to human immunoglobulinlabeled with horseradish peroxidase (conjugate) are added thereto, andthe plate is reincubated for 35 min at 37° C., then washed by theaforementioned buffer and distilled water. The reaction with conjugateis determined by adding chromogen, i.e., orthophenylenediamine in thepresence of 30% hydrogen peroxide. The intensity of color development isevaluated by using the rider (available Multiskan microplate rider) atthe 492 nm wavelength.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1) A method for aiding in assessing the risk that a human may besusceptible to stroke and/or transient ischemic attacks (TIA)comprising: a) obtaining a test sample of human blood, serum, plasma orother human body fluid from a human subject; b) analyzing the obtainedtest sample for the presence or amount of an NR2 antibody; and c)comparing the result of step (b) with a corresponding reference amountof an NR2 antibody, wherein the corresponding reference amount isderived from a population of apparently health human subjects. 2) Themethod of claim 1 wherein the presence or amount of an NR2A antibody andan NR2B antibody are measured. 3) The method of claim 1 wherein thepresence or amount of an NR2A antibody to the N-terminal domain of NR2A,and an NR2B antibody to the N-terminal domain of NR2B, are measured. 4)The method of claim 1 wherein the presence or amount of NR2 antibody ismeasured from the level of antibodies against a peptide of SEQ ID NO:2,3, 11, and/or 12, or a fragment, analogue, derivative or combinationthereof. 5) The method of claim 1 wherein the NR2 antibody is measuredby immunoassay. 6) The method of claim 1 wherein the NR2 antibody isdetected or measured by agglutination comprising: a) contacting the testsample with NR2 antigen bound on an agglutinating carrier for sufficienttime and under conditions to promote agglutination between the NR2antigen and NR2 antibody in said test sample; and b) reading a signalgenerated from the agglutination; wherein the signal correlates to thetiter of NR2 antibody present in the sample. 7) The method of claim 1,wherein the test sample is blood, urine, blood plasma, blood serum,cerebrospinal fluid, saliva, perspiration or brain tissue, or aderivative thereof. 8) The method of claim 1 wherein levels of NR2antibody are measured by a process comprising: a) contacting the testsample with a NR2 antigen capture agent for a time sufficient and underconditions to form a complex between said NR2 antigen capture agent andNR2 antibody in said test sample; b) contacting the complex with anindicator reagent comprising a secondary antibody attached to a signalgenerating compound; and c) measuring the signal. 9) A method for aidingin the differential diagnosis of ischemic versus hemorrhagic strokecomprising: a) obtaining a test sample from a human subject at the timesaid subject is suspected of suffering from an ischemic or hemorrhagicstroke; b) analyzing the obtained test sample for the presence or amountof an NR2 antigen or an NR2 antibody, or any combination thereof; and c)comparing the result of step (b) with a corresponding reference amountof an NR2 antigen or an NR2 antibody, or a combination thereof, whereinthe corresponding reference amount is derived from a population ofapparently health human subjects. 10) A method for aiding in thediagnosis that a human may be having a stroke and/or transient ischemicattack (TIA) comprising: a) obtaining a test sample of human blood,serum, plasma or other human body fluid from a human subject; b)analyzing the obtained test sample for the presence or amount of an NR2antigen; and c) comparing the result of step (b) with a correspondingreference amount of an NR2 antigen, wherein the corresponding referenceamount is derived from a population of apparently health human subjects.